MODIFIED NK-92 haNK003 CELLS FOR THE CLINIC

ABSTRACT

Provided herein are populations of modified NK-92 cells, compositions and kits comprising the cells, and methods of making and using the populations of cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/424,201, filed May 28, 2019, which is a divisional of U.S.application Ser. No. 15/914,665, filed Mar. 7, 2018, which claimspriority to U.S. Provisional Application No. 62/468,890, filed Mar. 8,2017, the contents of which are herein incorporated by reference intheir entirety for all purposes

BACKGROUND

Anticancer treatment with monoclonal antibodies (mAbs) has significantlyimproved the clinical outcome in patients with cancer. One of the majormechanisms of action of therapeutic antibodies is through antibodydependent cellular cytotoxicity (ADCC). Natural killer cells could beused as cytotoxic effector cells for cell-based immunotherapy since theyare a major effector cell for ADCC.

NK-92 is a cytolytic cancer cell line which was discovered in the bloodof a subject suffering from a non-Hodgkin's lymphoma and thenimmortalized ex vivo. NK-92 cells are derived from NK cells, but lackthe major inhibitory receptors that are displayed by normal NK cells,while retaining the majority of the activating receptors. NK-92 cells donot, however, attack normal cells nor do they elicit an unacceptableimmune rejection response in humans. Characterization of the NK-92 cellline is disclosed in WO 1998/49268 and U.S. Patent ApplicationPublication No. 2002-0068044. NK-92 cells have also been evaluated as apotential therapeutic agent in the treatment of certain cancers.

Although NK-92 cells retain almost all of the activating receptors andcytolytic pathways associated with NK cells, they do not express CD16 ontheir cell surfaces. CD16 is an Fc receptor which recognizes and bindsto the Fc portion of an antibody to activate NK cells for the ADCCeffector mechanism. Because they lack CD16 receptors, unmodified NK-92cells are unable to lyse target cells via the ADCC mechanism.

BRIEF SUMMARY

Provided herein are populations of modified NK-92 cells, compositionsand kits comprising the cells, and methods of making and using thepopulations of cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing haNK003 cell expansion. FIG. 1B is a graphshowing population doubling level (PDL) of haNK003 cells. Viability (%),cell density (cells/mL), and cumulative PDL were monitored over theexpansion period.

FIG. 2 shows representative histograms for the expression of surfacemarkers in aNK and haNK003 cells.

FIG. 3A is a graph showing natural cytotoxicity of haNK003 cells againstK562 cells.

FIG. 3B is a graph showing natural cytotoxicity of haNK003 cells againstRaji cells. FIG. 3C is a graph showing natural cytotoxicity of haNK003cells against SKOV3 cells. FIG. 3D is a graph showing naturalcytotoxicity of haNK003 cells against SKBR3 cells.

FIG. 4A is a graph showing ADCC of haNK003 cells against Raji cells.FIG. 4B is a graph showing ADCC of haNK003 cells against SKOV3 cells.FIG. 4C is a graph showing ADCC of haNK003 cells against SKBR3 cells.

FIG. 5 is a graph showing natural cytotoxic activity of irradiated vs.non-irradiated haNK003 cells against K562 cells. haNK003 cells weremock-irradiated (solid line) or irradiated at 10 Gy. Cytotoxic activityof irradiated haNK003 against K562 cells was assayed at 6 hr (dashedline) or 24 hr (dotted line) post-irradiation.

FIG. 6 is a graph showing natural cytotoxic activity of irradiated vs.non-irradiated haNK003 cells against DOHH2 cells. haNK003 cells weremock-irradiated (solid line) or irradiated at 10 Gy. Cytotoxic activityof irradiated haNK003 against DOHH2 cells was assayed at 6 hr (dashedline) or 24 hr (dotted line) post-irradiation. Note that data points forE:T ratios of 20:1 were not obtained for irradiated cells because celldeath resulted in insufficient numbers of haNK003 cells.

FIG. 7 is a graph showing ADCC activity of irradiated vs. non-irradiatedhaNK003 cells against DOHH2 cells. haNK003 cells were mock irradiated(solid symbols) or irradiated at 10 Gy (hollow symbols). ADCC activityof irradiated and non-irradiated haNK003 against DOHH2 cells was assayedat 6 hr (dashed lines) or 24 hr (dotted lines) post-irradiation, incombination with Rituxan (squares) or with Herceptin (triangles), whichdoes not react with DOHH2 cells. Note that the data point for E:T ratioof 20:1 was not obtained for irradiated cells at the 24 hour time pointbecause cell death resulted in insufficient numbers of haNK003 cells.

FIG. 8A is a graph showing IL-2 released (pg/mL) per 1×10⁶ cells ofirradiated vs. non-irradiated cells at 6, 12, 24 and 48 hours (h) asdetermined by sandwich ELISA run #1. FIG. 8B is a graph showing IL-2released (pg/mL) per 1×10⁶ cells of irradiated vs. non-irradiated cellsat 6, 12, 24 and 48 hours (h) as determined by sandwich ELISA run #2.FIG. 8C is a graph showing IL-2 released (pg/mL) per 1×10⁶ cells ofirradiated vs. non-irradiated cells at 6, 12, 24 and 48 hours (h) asdetermined by multiplex ELISA run #1. FIG. 8D is a graph showing IL-2released (pg/mL) per 1×10⁶ cells of irradiated vs. non-irradiated cellsat 6, 12, 24 and 48 hours (h) as determined by multiplex ELISA run #2.

FIG. 9A is a graph showing total intracellular IL-2 content (pg) per1×10⁶ cells of irradiated vs. non-irradiated cells at 6, 12, 24 and 48hours (h) as determined by sandwich ELISA run #1. FIG. 9B is a graphshowing total intracellular IL-2 content (pg) per 1×10⁶ cells ofirradiated vs. non-irradiated cells at 6, 12, 24 and 48 hours (h) asdetermined by sandwich ELISA run #2. FIG. 9C is a graph showing totalintracellular IL-2 content (pg) per 1×10⁶ cells of irradiated vs.non-irradiated cells at 6, 12, 24 and 48 hours (h) as determined bymultiplex ELISA run #1. FIG. 9D is a graph showing total intracellularIL-2 content (pg) per 1×10⁶ cells of irradiated vs. non-irradiated cellsat 6, 12, 24 and 48 hours (h) as determined by multiplex ELISA run #2.

FIG. 10A is a graph showing the amount of solubilized IL-2 (pg) per1×10⁶ cells in run #1 as determined by multiplex and sandwich ELISA.FIG. 10B is a graph showing the amount of solubilized IL-2 (pg) per1×10⁶ cells in run #2 as determined by multiplex and sandwich ELISA.

FIG. 11 is a graph showing the effect of haNK003 administeredintravenously on animal body weight in NOD/SCID mice. NOD/SCID mice (3male and 3 female per group) were treated by i.v. injection of PBS,non-irradiated or irradiated haNK003 cells at the dose of 1×10⁷ cells asa single dose, respectively. Animal body weight was monitored twiceweekly for 5 weeks. Values are mean±SEM, n=6.

FIG. 12 is a graph showing the effect of haNK003 cells on animal bodyweight in NOD/SCID mice. NOD/SCID mice (3 male and 3 female per group)were treated with PBS, 1×10⁷ of non-irradiated or irradiated haNK003cells once weekly for 4 weeks, animal body weight was monitored twiceweekly for 5 weeks. Values are mean±SEM, n=6.

FIG. 13 is a graph showing the comparison of aNK vs. haNK003 cells withrespect to natural cytotoxicity against K562 cells.

FIG. 14 is a graph showing the comparison of aNK vs. haNK003 cells withrespect to natural cytotoxicity against Daudi cells.

FIG. 15 is a graph showing the comparison of aNK vs. haNK003 cells withrespect to natural cytotoxicity against DOHH2 cells.

FIG. 16 is a graph showing the comparison of aNK vs. haNK003 cells withrespect to natural cytotoxicity against SKOV-3 cells.

FIG. 17 is a graph showing the comparison of aNK vs. haNK003 cells withrespect to natural cytotoxicity against HL-60 cells.

FIG. 18 is a graph showing the comparison of aNK vs. haNK003 cells withrespect to natural cytotoxicity against SR-91 cells.

FIG. 19 is a graph showing antitumor activity of haNK003 cells inMDA-MB-453 s.c. xenograft model in female NSG mice. Female NSG micebearing MDA-MB-453 human breast carcinoma tumors were treated by i.v.injection of PBS or irradiated haNK003 cells at the dose of 2.5×106 or1×107 cells twice weekly for four weeks, respectively. Tumor volumeswere monitored twice weekly. Values are mean±SEM; n=8.

FIG. 20 is a graph showing effect of haNK003 cells on animal body weightin female NSG mice. Female NSG mice bearing MDA-MB-453 human breastcarcinoma tumors were treated by i.v. injection of PBS or irradiatedhaNK003 cells at the dose of 2.5×106 or 1.0×107 cells twice weekly forfour weeks, respectively. Mice body weights were monitored twice weekly.Values are mean±SEM; n=4.

FIG. 21 is a table showing sample pairwise distances for gene expressionin normal NK cells 950, 962, and 996 as well as for haNK cells under 20%oxygen and 0% oxygen (hypoxic) conditions.

FIG. 22 is a table showing the genes exhibiting the most variability inexpression between 20% oxygen conditions and 0% oxygen conditions in950, 962, 996 and haNK cells.

FIG. 23 is a table showing the change in expression in the genesexhibiting the most change between 20% oxygen conditions and 0% oxygenconditions in 950, 962, 996 and haNK cells.

FIG. 24 is a table showing the change in expression in genes associatedwith hypoxia in haNK cells and NK cells 950, 962 and 996 between 20%oxygen conditions and 0% oxygen conditions.

FIG. 25 are graphs showing flow cytometric analysis of CD16 expressionin haNK003 cells and donor NK cells before and after PMA treatment. Downregulation of CD16 expression is 94.36%±3 in donor NK cells and 30%±0.04in haNK003 cells. aNK (NK-92 cells without CD16) were used as a control.

FIG. 26 are graphs showing flow cytometric analysis of CD16 expressionlevel in haNK003 cells and donor NK cells co-cultured with K562 cells(E:T=1:1). After 4 hours haNK003 cells showed stable CD16 expression incomparison to donor NK cells. The CD16 down regulation in donor NK cellsafter 4 hour of co-culture with K562 was 60.25%±09 and 4.9%±2.57 forhaNK003 cells. After overnight recovery, there was still a 57.54%±26.82downregulation of CD16 expression in donor NK cells, whereas in haNK003cells the CD16 level recovered to close to normal, with only 2.78%±3.5of down regulation. aNK (NK-92 cells without CD16) were used as acontrol.

FIGS. 27A and 27B are graphs showing CD16 expression level in haNK003cells after ADCC. ADCC was performed by co-culturing haNK003 cells andDoHH in presence of 1 μg/ml Rituximab for 4 hours at E:T ratio of 1:0(effectors alone) to 1:4. CD16 expression level was measured at 4 hoursand after 24 hours by flow cytometry. FIG. 27A shows flow cytometricanalysis of CD16 expression level in haNK-003 after ADCC along withcontrol (E:T=1:0). FIG. 27B shows median fluorescence intensity (MFI) ofCD16 expression after ADCC and 24 hours after ADCC.

DETAILED DESCRIPTION

Provided herein are modified NK-92 haNK003 cells. The cells express theFc Receptor CD16 and an endoplasmic reticulum bound form of IL-2. Thus,the cells are not dependent on external IL-2 for growth. Further, themodified NK-92 cells have enhanced cytotoxic capabilities with theinsertion of the high affinity variant of the CD16 receptor, and aretherefore capable of CD16 targeted antibody-dependent cell-mediatedcytotoxicity (ADCC). ADCC is mediated by recognition of the Fc fragmentof the target-bound antibody (IgG) via the CD16 Fc receptor. Therefore,for oncological applications, ADCC by the modified cells is elicited byCD16 receptor binding to the Fc fragment of tumor cell-bound IgG, thusactivating the modified NK-92 cells for targeted killing. As describedherein, the provided modified NK-92 cells were created through stabletransfection with a bicistronic plasmid based vector containingsequences for CD16, the high affinity Fc-gamma receptor(FcγRIIIa/CD16a), as well as IL-2 that is targeted to the endoplasmicreticulum. The cells contain a plasmid sequence that was inserted at asingle location on Chromosome 17 at position 15,654,977 on the + strand.The modified NK-92 cells produce endogenous IL-2 and are phenotypicallyCD56+, CD3−, and CD16+. The modified NK-92 haNK003 cells provided in thepresent application are sometimes referred to herein as simply haNK003cells.

As described in more detail in the examples below, NK-92 cells weretransformed with the pNEUKv1_FcRIL2 plasmid (SEQ ID NO:1). ThepNEUKv1_FcRIL2 plasmid is a bicistronic construct expressing a modifiedCD16 that contains a valine at amino acid 176 (when referring to thefull length CD16 peptide) and IL-2 with an endoplasmic reticulumretention signal. Whole genome sequencing (WGS) of the cells wereperformed, resulting in identification of one plasmid insertion site atChromosome 17. WGS confirmed that the integration of the bicistronicplasmid in the haNK003 cell line is in a region of the genome that isdistant from any gene with oncogenic potential. The nearest 5′ geneTBC1D26 is 10,722 bp upstream, and the nearest 3′ gene ADORA2B is186,828 bp downstream. The modified NK-92 cells grow consistently whenpassaged every 3 to 4 days and seeded at a density of approximately0.3-0.5×10⁶ cells/mL. The mean doubling time was 65 (48-95) hours fromday 3 to day 29. Analysis of flow cytometry data shows that modifiedNK-92 cells express CD54, CD56, NKG2D, NKp30, and CD16 surface markerproteins and lack CD3. The modified NK-92 cells are capable of growingwithout supplementation of IL-2 in the culture media. Further, it wasdetermined that the modified NK-92 cells expressing IL-2 release lowlevels of IL-2 into culture media. Non-irradiated haNK003 cells alonesecrete on average approximately 276.1 pg/mL per 1,000,000 cells at 6hours and up to 1403.3 pg/mL per 1,000,000 cells at 48 hours in culture.The provided modified NK-92 cells are naturally cytotoxic to severalcancer cell lines and are capable of enhanced specific lysis via ADCCwhen combined with antibodies.

The NK-92 cell line was discovered to proliferate in the presence ofinterleukin 2 (IL-2). Gong et al., Leukemia 8:652-658 (1994). Thesecells have high cytolytic activity against a variety of cancers. TheNK-92 cell line is a homogeneous NK cell population having broadanti-tumor cytotoxicity with predictable yield after expansion. Phase Iclinical trials have confirmed its safety profile. NK-92 was discoveredin the blood of a subject suffering from a non-Hodgkin's lymphoma andthen immortalized ex vivo. NK-92 cells are derived from NK cells, butlack the major inhibitory receptors that are displayed by normal NKcells, while retaining the majority of the activating receptors. NK-92cells do not, however, attack normal cells nor do they elicit anunacceptable immune rejection response in humans. Characterization ofthe NK-92 cell line is disclosed in WO 1998/49268 and U.S. PatentApplication Publication No. 2002-0068044.

NK-92 cells are known and include, but are not limited to, thosedescribed in, e.g., U.S. Pat. Nos. 7,618,817, 8,034,332, and 8,313,943,US Patent Application Publication No. 2013/0040386, all of which areincorporated herein by reference in their entireties, such as wild typeNK-92, NK-92-CD16, NK-92-CD16-γ, NK-92-CD16-ζ, NK-92-CD16(F176V),NK-92MI and NK-92CI.

Provided herein is a population of modified NK-92 haNK003 cells havingantibody-dependent cell-mediated cytotoxicity (ADCC) comprising nucleicacid molecules comprising both CD16 (SEQ ID NO:3) and IL-2 (SEQ IDNO:5), wherein greater than 90% of the cells in the population of cellsexpress CD56, CD16, CD54, and NKp30 and less than 5% of the cells in thepopulation of cells express CD3. Optionally, the nucleic acid moleculesare mRNA molecules. Optionally, the mRNA molecules comprise from 5′ to3′ a sequence encoding CD16, an IRES sequence, and a sequence encodingIL-2. Optionally, the cells comprise SEQ ID NO:1 on chromosome 17.Optionally, the mean doubling time of the cells is between 55 and 70hours. Optionally, the population of cells maintains the mean doublingtime from 1, 2, 3, 4, 5, 10, 15, 20, 25 or more days. Optionally, thepopulation of cells can be passaged for 1, 2, 3, 4 or more days.Optionally, the cells secrete IL-2 at a concentration of 10 to 40pg/hour per million cells. Optionally, the cells are irradiated cells.

In response to certain stimuli, CD16 is cleaved close to the cellmembrane resulting in release of the extracellular portion of thereceptor and down regulation of expression following activation (See,Jing, et al., PLOS one, 10(3):e0121788 DOI:10.1371/journal.pone.0121788(2015)). Under normal conditions, this mechanism helps to control NKcell cytotoxicity, but in the tumor environment, this can reduce ADCCpotency and cancer cell killing. Advantageously, the provided haNK003cells have enhanced ADCC activity against cancer cells. Without beingbound by theory, this is believed to be due to the stable expression ofCD16 in haNK003 cells event after ADCC. As shown in the examples below,after activation with phorbol-12-myristate 13-acetate or stimulationwith K562 cells, expression of CD16 remained high as compared to controlcells. Further, CD16 expression remained high in haNK003 cells evenafter ADCC. Therefore, the provided haNK003 cells have reduceddownregulation of expression of CD16 compared to a control. Further, thehaNK003 cells have increased levels of CD16 after ADCC compared to acontrol. Stated another way, the cells maintain higher levels of CD16after ADCC compared to a control. Thus, haNK003 cells have more stableexpression of CD16 compared to a control, e.g., normal NK cells.

Natural Killer (NK) cell lytic activity is suppressed in hypoxicenvironments in vitro (1% O₂) and is associated with downregulation ofNKG2D, perforin and granzyme. There is some variability with NKsensitivity to hypoxia (1% O₂) from normal donors. However, NK celllytic activity can be partially rescued by exogenous IL-2 activation invitro (16 h, 1000 IU/ml). Further, NK cells retain ADCC capacity atunder 1% oxygen conditions. As described in more detail in the examplesbelow, genes associated with hypoxia show no change in expression inhaNK cells between 20% oxygen conditions and 0% oxygen (hypoxic)conditions. However, these same genes associated with hypoxia are shownto have reduced expression in normal NK cells.

As noted above, the modified NK-92 cells express the Fc receptor CD16.As used herein, the term “Fc receptor” refers to a protein found on thesurface of certain cells (e.g., natural killer cells) that contribute tothe protective functions of the immune cells by binding to part of anantibody known as the Fc region. Binding of the Fc region of an antibodyto the Fc receptor (FcR) of a cell stimulates phagocytic or cytotoxicactivity of a cell via antibody-mediated phagocytosis orantibody-dependent cell-mediated cytotoxicity (ADCC). FcRs areclassified by the type of antibody they recognize. For example, Fc-gammareceptors (FCγR) bind to the IgG class of antibodies. FCγRIII-A (alsocalled CD16) is a low affinity Fc receptor that binds to IgG antibodiesand activates ADCC. FCγRIII-A are typically found on NK cells. Arepresentative amino acid sequence encoding CD16 is shown in SEQ IDNO:3. A representative polynucleotide sequence encoding CD16 is shown inSEQ ID NO:4. The complete sequences of CD16 can be found in theSwissProt database as entry P08637.

Optionally, the modified NK-92 cells comprise a nucleic acid sequencewith 70%, 80%, 90%, or 95% identity to SEQ ID NO:3. Optionally, themodified NK-92 cells comprise a nucleic acid sequence with 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3.Optionally, the modified NK-92 cells comprise a polypeptide with 70%,80%, 90%, or 95% identity to SEQ ID NO:4. Optionally, the modified NK-92cells comprise a polypeptide with 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identity to SEQ ID NO:4.

The cytotoxicity of NK-92 cells is dependent on the presence ofcytokines (e.g., interleukin-2 (IL-2)). The cost of using exogenouslyadded IL-2 needed to maintain and expand NK-92 cells in commercial scaleculture is significant. The administration of IL-2 to human subjects insufficient quantity to continue activation of NK-92 cells would causeadverse side effects. Optionally, the IL-2 is expressed with a signalsequence that directs the IL-2 to the endoplasmic reticulum. Directingthe IL-2 to the endoplasmic reticulum permits expression of IL-2 atlevels sufficient for autocrine activation and without releasingsubstantial amounts of IL-2 extracellularly. See Konstantinidis et al“Targeting IL-2 to the endoplasmic reticulum confines autocrine growthstimulation to NK-92 cells” Exp Hematol. 2005 February; 33(2):159-64. Arepresentative nucleic acid encoding IL-2 is shown in SEQ ID NO:5 and arepresentative polypeptide of IL-2 is shown in SEQ ID NO:6.

Optionally, the modified NK-92 cells comprise a nucleic acid sequencewith 70%, 80%, 90%, or 95% identity to SEQ ID NO:5. Optionally, themodified NK-92 cells comprise a nucleic acid sequence with 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5.Optionally, the modified NK-92 cells comprise a polypeptide with 70%,80%, 90%, or 95% identity to SEQ ID NO:6. Optionally, the modified NK-92cells comprise a polypeptide with 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identity to SEQ ID NO:6. The provided modified NK-92cells advantageously are capable of being maintained in the absence ofIL-2 without secreting IL-2 in an amount to cause a clinical adverseeffect.

Nucleic acid, as used herein, refers to deoxyribonucleotides orribonucleotides and polymers and complements thereof. The term includesdeoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form. The term encompasses nucleic acids containingknown nucleotide analogs or modified backbone residues or linkages,which are synthetic, naturally occurring, and non-naturally occurring,which have similar binding properties as the reference nucleic acid, andwhich are metabolized in a manner similar to the reference nucleotides.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unlessotherwise indicated, conservatively modified variants of nucleic acidsequences (e.g., degenerate codon substitutions) and complementarysequences can be used in place of a particular nucleic acid sequencerecited herein. Specifically, degenerate codon substitutions may beachieved by generating sequences in which the third position of one ormore selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al.,Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

A nucleic acid is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA thatencodes a presequence or secretory leader is operably linked to DNA thatencodes a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, operably linked means that the DNA sequences being linked arenear each other, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous. Forexample, a nucleic acid sequence that is operably linked to a secondnucleic acid sequence is covalently linked, either directly orindirectly, to such second sequence, although any effectivethree-dimensional association is acceptable. A single nucleic acidsequence can be operably linked to multiple other sequences. Forexample, a single promoter can direct transcription of multiple RNAspecies. Linking can be accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

The terms identical or percent identity, in the context of two or morenucleic acids or polypeptide sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same (i.e., about 60%identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher identity over a specified region,when compared and aligned for maximum correspondence over a comparisonwindow or designated region) as measured using a BLAST or BLAST 2.0sequence comparison algorithms with default parameters described below,or by manual alignment and visual inspection (see, e.g., NCBI web siteor the like). Such sequences are then said to be substantiallyidentical. This definition also refers to, or may be applied to, thecompliment of a test sequence. The definition also includes sequencesthat have deletions and/or additions, as well as those that havesubstitutions. As described below, the preferred algorithms can accountfor gaps and the like. Preferably, identity exists over a region that isat least about 25 amino acids or nucleotides in length, or morepreferably over a region that is 50-100 amino acids or nucleotides inlength.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer; subsequence coordinates are designated, if necessary; andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A comparison window, as used herein, includes reference to a segment ofany one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981); by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970);by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988); by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.); or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977), and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for nucleicacids or proteins. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information, asknown in the art. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of a selected length(W) in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated for nucleotide sequencesusing the parameters M (reward score for a pair of matching residues;always >0) and N (penalty score for mismatching residues; always <0).For amino acid sequences, a scoring matrix is used to calculate thecumulative score. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The Expectation value (E) represents the number ofdifferent alignments with scores equivalent to or better than what isexpected to occur in a database search by chance. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)),alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The term polypeptide, as used herein, generally has its art-recognizedmeaning of a polymer of at least three amino acids and is intended toinclude peptides and proteins. However, the term is also used to referto specific functional classes of polypeptides, such as, for example,desaturases, elongases, etc. For each such class, the present disclosureprovides several examples of known sequences of such polypeptides. Thoseof ordinary skill in the art will appreciate, however, that the termpolypeptide is intended to be sufficiently general as to encompass notonly polypeptides having the complete sequence recited herein (or in areference or database specifically mentioned herein), but also toencompass polypeptides that represent functional fragments (i.e.,fragments retaining at least one activity) of such completepolypeptides. Moreover, those in the art understand that proteinsequences generally tolerate some substitution without destroyingactivity. Thus, any polypeptide that retains activity and shares atleast about 30-40% overall sequence identity, often greater than about50%, 60%, 70%, or 80%, and further usually including at least one regionof much higher identity, often greater than 90% or even 95%, 96%, 97%,98%, or 99% in one or more highly conserved regions, usuallyencompassing at least 3-4 and often up to 20 or more amino acids, withanother polypeptide of the same class, is encompassed within therelevant term polypeptide as used herein. Those in the art can determineother regions of similarity and/or identity by analysis of the sequencesof various polypeptides described herein. As is known by those in theart, a variety of strategies are known and tools are available forperforming comparisons of amino acid or nucleotide sequences to assessdegrees of identity and/or similarity. These strategies include, forexample, manual alignment, computer assisted sequence alignment andcombinations thereof. A number of algorithms (which are generallycomputer implemented) for performing sequence alignment are widelyavailable, or can be produced by one of skill in the art. Representativealgorithms include, e.g., the local homology algorithm of Smith andWaterman (Adv. Appl. Math., 1981, 2: 482); the homology alignmentalgorithm of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443); thesearch for similarity method of Pearson and Lipman (Proc. Natl. Acad.Sci. (USA), 1988, 85: 2444); and/or by computerized implementations ofthese algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package Release 7.0, Genetics Computer Group, 575Science Dr., Madison, Wis.). Readily available computer programsincorporating such algorithms include, for example, BLASTN, BLASTP,Gapped BLAST, PILEUP, CLUSTALW, etc. When utilizing BLAST and GappedBLAST programs, default parameters of the respective programs may beused. Alternatively, the practitioner may use non-default parametersdepending on his or her experimental and/or other requirements (see forexample, the Web site having URL www.ncbi.nlm.nih.gov).

As used herein, the terms promoter, promoter element, and regulatorysequence refer to a polynucleotide that regulates expression of aselected polynucleotide sequence operably linked to the promoter, andthat effects expression of the selected polynucleotide sequence incells.

The term transformation as used herein refers to a process by which anexogenous or heterologous nucleic acid molecule (e.g., a vector orrecombinant nucleic acid molecule) is introduced into a recipient cellor microorganism. The exogenous or heterologous nucleic acid moleculemay or may not be integrated into (i.e., covalently linked to)chromosomal DNA making up the genome of the host cell or microorganism.For example, the exogenous or heterologous polynucleotide may bemaintained on an episomal element, such as a plasmid. Alternatively oradditionally, the exogenous or heterologous polynucleotide may becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosomal replication. Methods for transformation include, butare not limited to, calcium phosphate precipitation; fusion of recipientcells with bacterial protoplasts containing the recombinant nucleicacid; treatment of the recipient cells with liposomes containing therecombinant nucleic acid; DEAE dextran; fusion using polyethylene glycol(PEG); electroporation; magnetoporation; biolistic delivery; retroviralinfection; lipofection; and micro-injection of DNA directly into cells.

The term transformed, as used in reference to cells, refers to cellsthat have undergone transformation as described herein such that thecells carry exogenous or heterologous genetic material (e.g., arecombinant nucleic acid). The term transformed can also oralternatively be used to refer to microorganisms, strains ofmicroorganisms, tissues, organisms, etc. that contain exogenous orheterologous genetic material.

The terms modified and recombinant when used with reference to a cell,nucleic acid, polypeptide, vector, or the like indicates that the cell,nucleic acid, polypeptide, vector or the like has been modified by or isthe result of laboratory methods and is non-naturally occurring. Thus,for example, modified cells include cells produced by or modified bylaboratory methods, e.g., transformation methods for introducing nucleicacids into the cell. Modified cells can include nucleic acid sequencesnot found within the native (non-recombinant) form of the cells or caninclude nucleic acid sequences that have been altered, e.g., linked to anon-native promoter.

As described herein, a control or standard control refers to a sample,measurement, or value that serves as a reference, usually a knownreference, for comparison to a test sample, measurement, or value. Forexample, a test cell, e.g., a cell transformed with nucleic acidsequences encoding genes for an Fc Receptor can be compared to a knownnormal (wild-type) cell (e.g., a standard control cell). A standardcontrol can also represent an average measurement or value gathered froma population of cells (e.g., standard control microorganisms) that donot express the Fc Receptor or that do not have or have minimal levelsof Fc Receptor activity. One of skill will recognize that standardcontrols can be designed for assessment of any number of parameters(e.g., RNA levels, polypeptide levels, specific cell types, and thelike).

As used herein, the term “antibody” refers to an immunoglobulin orfragment thereof. The antibody may be of any type (e.g., IgG, IgA, IgM,IgE or IgD). Preferably, the antibody is IgG. An antibody may benon-human (e.g., from mouse, goat, or any other animal), fully human,humanized, or chimeric. An antibody may be polyclonal or monoclonal.Optionally, the antibody is monoclonal.

The term “monoclonal antibody” as used herein, refers to a pure,target-specific antibody produced from a single clone of cells grown inculture and that is capable of indefinitely proliferating. Monoclonalantibodies that may be used include naked antibodies, that attach to andblock antigens on cancerous cells. Optionally, the naked monoclonalantibody is alemtuzumab, which binds to the CD52 antigen in lymphocytes.Also included in the monoclonal antibodies that may be used areconjugated monoclonal antibodies, such as tagged, labeled, or loadedantibodies. Specifically, the antibodies may be tagged or loaded with adrug or a toxin, or radioactively labeled. Examples of such antibodiesinclude, but are not limited to, ibritumomab, which targets the CD20antigen; brentuximab, which targets the CD30 antigen, and trastuzumab,which targets the HER2 protein. Other monoclonal antibodies that may beused are bispecific monoclonal antibodies, such as blinatunomab, whichtargets CD19 in lymphoma cells, and CD3 in T cells.

As used herein, the term “antibody fragment” refers to any portion ofthe antibody that recognizes an epitope. Antibody fragments may beglycosylated. By way of non-limiting example, the antibody fragment maybe a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fv fragment,an rIgG fragment, a functional antibody fragment, single chainrecombinant forms of the foregoing, and the like. F(ab′)2, Fab, Fab′ andFv are antigen-binding fragments that can be generated from the variableregion of IgG and IgM. They vary in size, valency, and Fc content. Thefragments may be generated by any method, including expression of theconstituents (e.g., heavy and light chain portions) by a cell or cellline, or multiple cells or cell lines. Preferably, the antibody fragmentrecognizes the epitope and contains a sufficient portion of an Fc regionsuch that it is capable of binding an Fc receptor.

As used herein, the term “cancer” refers to all types of cancer,neoplasm, or malignant tumors found in mammals, including leukemia,carcinomas and sarcomas. Exemplary cancers include cancer of the brain,breast, cervix, colon, head & neck, liver, kidney, lung, non-small celllung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus andmedulloblastoma. Additional examples include, Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, ovarian cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,primary brain tumors, cancer, malignant pancreatic insulanoma, malignantcarcinoid, urinary bladder cancer, premalignant skin lesions, testicularcancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,genitourinary tract cancer, malignant hypercalcemia, endometrial cancer,adrenal cortical cancer, neoplasms of the endocrine and exocrinepancreas, and prostate cancer.

Also provided are methods of treating subjects with modified NK-92 cellsas described herein. Optionally, the subject is treated with themodified NK-92 cell and an antibody.

Modified NK-92 cells can be administered to a subject by absolutenumbers of cells, e.g., said subject can be administered from about 1000cells/injection to up to about 10 billion cells/injection, such as atabout, at least about, or at most about, 1×10¹⁰, 1×10⁹, 1×10⁸, 1×10⁷,5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴, 1×10³, 5×10³ (and soforth) NK-92 cells per injection, or any ranges between any two of thenumbers, end points inclusive. Optionally, from 1×10⁸ to 1×10¹⁰ cellsare administered to the subject. Optionally, the cells are administeredone or more times weekly for one or more weeks. Optionally, the cellsare administered once or twice weekly for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more weeks.

Optionally, subject are administered from about 1000 cells/injection/m²to up to about 10 billion cells/injection/m², such as at about, at leastabout, or at most about, 1×10⁸/m², 1×10⁷/m², 5×10⁷/m², 1×10⁶/m²,5×10⁶/m², 1×10⁵/m², 5×10⁵/m², 1×10⁴/m², 5×10⁴/m², 1×10³/m², 5×10³/m²(and so forth) NK-92 cells per injection, or any ranges between any twoof the numbers, end points inclusive.

Optionally, NK-92 cells can be administered to such individual byrelative numbers of cells, e.g., said individual can be administeredabout 1000 cells to up to about 10 billion cells per kilogram of theindividual, such as at about, at least about, or at most about, 1×10⁸,1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴, 1×10³, 5×10³(and so forth) NK-92 cells per kilogram of the individual, or any rangesbetween any two of the numbers, end points inclusive.

Optionally, the total dose may calculated by m² of body surface area,including about 1×10¹¹, 1×10¹⁰, 1×10⁹, 1×10⁸, 1×10⁷, per m², or anyranges between any two of the numbers, end points inclusive. Optionally,between about 1 billion and about 3 billion NK-92 cells are administeredto a patient. Optionally, the amount of NK-92 cells injected per dosemay calculated by m2 of body surface area, including 1×10¹¹, 1×10¹⁰,1×10⁹, 1×10⁸, 1×10⁷, per m².

The NK-92 cells, and optionally other anti-cancer agents can beadministered once to a patient with cancer can be administered multipletimes, e.g., once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22 or 23 hours, or once every 1, 2, 3, 4, 5,6 or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeksduring therapy, or any ranges between any two of the numbers, end pointsinclusive.

Optionally, NK-92 cells are administered in a composition comprisingNK-92 cells and a medium, such as human serum or an equivalent thereof.Optionally, the medium comprises human serum albumin. Optionally, themedium comprises human plasma. Optionally, the medium comprises about 1%to about 15% human serum or human serum equivalent.

Optionally, the medium comprises about 1% to about 10% human serum orhuman serum equivalent. Optionally, the medium comprises about 1% toabout 5% human serum or human serum equivalent. Optionally, the mediumcomprises about 2.5% human serum or human serum equivalent. Optionally,the serum is human AB serum. Optionally, a serum substitute that isacceptable for use in human therapeutics is used instead of human serum.Such serum substitutes may be known in the art. Optionally, NK-92 cellsare administered in a composition comprising NK-92 cells and an isotonicliquid solution that supports cell viability. Optionally, NK-92 cellsare administered in a composition that has been reconstituted from acryopreserved sample.

According to the methods provided herein, the subject is administered aneffective amount of one or more of the agents provided herein. The termseffective amount and effective dosage are used interchangeably. The termeffective amount is defined as any amount necessary to produce a desiredphysiologic response (e.g., reduction of inflammation). Effectiveamounts and schedules for administering the agent may be determinedempirically by one skilled in the art. The dosage ranges foradministration are those large enough to produce the desired effect inwhich one or more symptoms of the disease or disorder are affected(e.g., reduced or delayed). The dosage should not be so large as tocause substantial adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex, type of disease, theextent of the disease or disorder, route of administration, or whetherother drugs are included in the regimen, and can be determined by one ofskill in the art. The dosage can be adjusted by the individual physicianin the event of any contraindications. Dosages can vary and can beadministered in one or more dose administrations daily, for one orseveral days. Guidance can be found in the literature for appropriatedosages for given classes of pharmaceutical products. For example, forthe given parameter, an effective amount will show an increase ordecrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%,90%, or at least 100%. Efficacy can also be expressed as “-fold”increase or decrease. For example, a therapeutically effective amountcan have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effectover a control. The exact dose and formulation will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques (see, e.g., Lieberman, PharmaceuticalDosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical Compounding (1999); Remington: The Science andPractice of Pharmacy, 22nd Edition, Gennaro, Editor (2012), and Pickar,Dosage Calculations (1999)).

Pharmaceutically acceptable compositions can include a variety ofcarriers and excipients. A variety of aqueous carriers can be used,e.g., buffered saline and the like. These solutions are sterile andgenerally free of undesirable matter. Suitable carriers and excipientsand their formulations are described in Remington: The Science andPractice of Pharmacy, 21st Edition, David B. Troy, ed., LippicottWilliams & Wilkins (2005). By pharmaceutically acceptable carrier ismeant a material that is not biologically or otherwise undesirable,i.e., the material is administered to a subject without causingundesirable biological effects or interacting in a deleterious mannerwith the other components of the pharmaceutical composition in which itis contained. If administered to a subject, the carrier is optionallyselected to minimize degradation of the active ingredient and tominimize adverse side effects in the subject. As used herein, the termpharmaceutically acceptable is used synonymously with physiologicallyacceptable and pharmacologically acceptable. A pharmaceuticalcomposition will generally comprise agents for buffering andpreservation in storage and can include buffers and carriers forappropriate delivery, depending on the route of administration.

The compositions may contain acceptable auxiliary substances as requiredto approximate physiological conditions such as pH adjusting andbuffering agents, toxicity adjusting agents and the like, for example,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like. The concentration of cells in theseformulations and/or other agents can vary and will be selected primarilybased on fluid volumes, viscosities, body weight and the like inaccordance with the particular mode of administration selected and thesubject's needs.

Optionally, the NK-92 cells are administered to the subject inconjunction with one or more other treatments for the cancer beingtreated. Without being bound by theory, it is believed that co-treatmentof a subject with NK-92 cells and another therapy for the cancer willallow the NK-92 cells and the alternative therapy to give the endogenousimmune system a chance to clear the cancer that heretofore hadoverwhelmed such endogenous action. Optionally, two or more othertreatments for the cancer being treated includes, for example, anantibody, radiation, chemotherapeutic, stem cell transplantation, orhormone therapy.

Optionally, an antibody is administered to the patient in conjunctionwith the NK-92 cells. Optionally, the NK-92 cells and an antibody areadministered to the subject together, e.g., in the same formulation;separately, e.g., in separate formulations, concurrently; or can beadministered separately, e.g., on different dosing schedules or atdifferent times of the day. When administered separately, the antibodycan be administered in any suitable route, such as intravenous or oraladministration.

Optionally, antibodies may be used to target cancerous cells or cellsthat express cancer-associated markers. A number of antibodies have beenapproved for the treatment of cancer, alone.

TABLE 2 Example FDA approved therapeutic monoclonal antibodies BrandIndication Antibody name Company Target (Targeted disease) AlemtuzumabCampath ® Genzyme CD52 Chronic lymphocytic leukemia BrentuximabAdcetris ® CD30 Anaplastic large cell vedotin lymphoma (ALCL) andHodgkin lymphoma Cetuximab Erbitux ® Bristol-Myers epidermal growthColorectal cancer, Head and Squibb/Eli factor receptor neck cancerLilly/Merck KGaA Gemtuzumab Mylotarg ® Wyeth CD33 Acute myelogenousleukemia (with calicheamicin) Ibritumomab Zevalin ® Spectrum CD20Non-Hodgkin tiuxetan Pharmaceuticals, lymphoma (with yttrium- Inc. 90 orindium-111) Ipilimumab (MD Yervoy ® blocks CTLA-4 Melanoma X-101)Ofatumumab Arzerra ® CD20 Chronic lymphocytic leukemia PalivizumabSynagis ® MedImmune an epitope of the Respiratory Syncytial Virus RSV Fprotein Panitumumab Vectibix ® Amgen epidermal growth Colorectal cancerfactor receptor Rituximab Rituxan ®, Biogen CD20 Non-Hodgkin lymphomaMabthera ® Idec/Genentech Tositumomab Bexxar ® GlaxoSmithKline CD20Non-Hodgkin lymphoma Trastuzumab Herceptin ® Genentech ErbB2 Breastcancer Blinatunomab bispecific CD19- Philadelphia directed CD3 T-cellchromosome-negative engager relapsed or refractory B cell precursoracute lymphoblastic leukemia (ALL) Avelumamab anti-PD-L1 Non-small celllung cancer, metastatic Merkel cell carcinoma; gastic cancer, breastcancer, ovarian cancer, bladder cancer, melanoma, meothelioma, includingmetastatic or locally advanced solid tumors Daratumumab CD38 Multiplemyeloma Elotuzumab a SLAMF7-directed Multiple myeloma (also known as CD319) immunostimulatory antibody

Antibodies may treat cancer through a number of mechanisms. ADCC occurswhen immune cells, such as NK cells, bind to antibodies that are boundto target cells through Fc receptors, such as CD16.

Accordingly, NK-92 cells that express CD16 are administered to a subjectalong with an effective amount of at least one monoclonal antibodydirected against a specific cancer-associated protein, for example,alemtuzumab, bevacizumab, ibritumomab tiuxetan, ofatumumab, rituximab,and trastuzumab. Optionally, the monoclonal antibody is a nakedmonoclonal antibody, a conjugated monoclonal antibody or a bispecificmonoclonal antibody. Optionally, a bispecific antibody can be used thatbinds the cancer cell and also binds a cell-surface protein present onthe surface of NK-92 cells.

Cancer-specific antibodies bind to particular protein antigens that areexpressed on the surfaces of cancer cells. NK-92 cells can be modifiedsuch that an antibody is associated with the NK-92 cell surface.Optionally, the antibody is specific for the cancer. In this way, theNK-92 cell can be specifically targeted to the cancer. Neutralizingantibodies may also be isolated. For example, a secreted glycoprotein,YKL-40, is elevated in multiple types of advanced human cancers. It iscontemplated that an antibody to YKL-40 could be used to restrain tumorgrowth, angiogenesis and/or metastasis. See Faibish et al., (2011) Mol.Cancer Ther. 10(5):742-751.

Antibodies to cancer can be purchased from commercially availablesources or can be produced by any method known in the art. For example,antibodies can be produced by obtaining B cells, bone marrow, or othersamples from previously one or more patients who were infected by thecancer and recovered or were recovering when the sample was taken.Methods of identifying, screening, and growing antibodies (e.g.,monoclonal antibodies) from these samples are known. For example, aphage display library can be made by isolating RNA from the sample orcells of interest, preparing cDNA from the isolated RNA, enriching thecDNA for heavy-chain and/or light-chain cDNA, and creating librariesusing a phage display vector. Libraries can be prepared and screened asdescribed, for example, in Maruyama, et al., which is incorporatedherein by reference in its entirety. Antibodies can be made byrecombinant methods or any other method. Isolation, screening,characterization, and production of human monoclonal antibodies are alsodescribed in Beerli, et al., PNAS (2008) 105(38):14336-14341, which isincorporated herein by reference in its entirety.

Combinations of agents or compositions can be administered eitherconcomitantly (e.g., as a mixture), separately but simultaneously (e.g.,via separate intravenous lines) or sequentially (e.g., one agent isadministered first followed by administration of the second agent).Thus, the term combination is used to refer to concomitant,simultaneous, or sequential administration of two or more agents orcompositions. The course of treatment is best determined on anindividual basis depending on the particular characteristics of thesubject and the type of treatment selected. The treatment, such as thosedisclosed herein, can be administered to the subject on a daily, twicedaily, bi-weekly, monthly, or any applicable basis that istherapeutically effective. The treatment can be administered alone or incombination with any other treatment disclosed herein or known in theart. The additional treatment can be administered simultaneously withthe first treatment, at a different time, or on an entirely differenttherapeutic schedule (e.g., the first treatment can be daily, while theadditional treatment is weekly).

Also disclosed are kits comprising the provided modified NK-92 cells.Optionally, the kits further include one or more additional agents suchas antibodies. The components of the kit may be contained in one ordifferent containers such as one or more vials. The antibody may be inliquid or solid form (e.g., after lyophilization) to enhance shelf-life.If in liquid form, the components may comprise additives such asstabilizers and/or preservatives such as proline, glycine, or sucrose orother additives that enhance shelf-life.

Optionally, the kit may contain additional compounds such astherapeutically active compounds or drugs that are to be administeredbefore, at the same time, or after administration of the modified NK-92cells or NK-92 cells and antibody. Examples of such compounds includevitamins, minerals, fludrocortisone, ibuprofen, lidocaine, quinidine,chemotherapeutic, and the like.

Optionally, instructions for use of the kits will include directions touse the kit components in the treatment of a cancer. The instructionsmay further contain information regarding how to prepare (e.g., diluteor reconstitute, in the case of freeze-dried protein) the antibody andthe NK-92 cells (e.g., thawing and/or culturing). The instructions mayfurther include guidance regarding the dosage and frequency ofadministration.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed while, specific references to each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a method is disclosed and discussed and a numberof modifications that can be made to a number of molecules including themethod are discussed, each and every combination and permutation of themethod and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Likewise,any subset or combination of these is also specifically contemplated anddisclosed. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods using the disclosedcompositions. Thus, if there are a variety of additional steps that canbe performed, it is understood that each of these additional steps canbe performed with any specific method steps or combination of methodsteps of the disclosed methods, and that each such combination or subsetof combinations is specifically contemplated and should be considereddisclosed.

Publications cited herein and the material for which they are cited arehereby specifically incorporated by reference in their entireties.

The examples below are intended to further illustrate certain aspects ofthe methods and compositions described herein, and are not intended tolimit the scope of the claims.

EXAMPLES Example 1. Structural and Functional Characteristics of haNK003

NK-92 [CD16.176V, ER IL-2] (haNK003) was generated through themodification of NK-92 cells. NK-92 cells were originally isolated in1992 from a 50-year-old male patient with rapidly progressivenon-Hodgkin's lymphoma (Gong, et al., Leukemia, 8(4):652-8 (1994)). TheNK-92 cell line was subsequently characterized and shown to bephenotypically CD56+, CD3-, and CD16-, as well as IL-2 dependent.haNK003 is an allogeneic cell line that was created through stabletransfection by electroporation of NK-92 cells with a bicistronicplasmid-based vector containing sequences for CD16 and IL-2. Thetransfected plasmid is shown in FIG. 1 and was constructed by GeneArtAG. The CD16 sequence codes for a valine at amino acid 176 (176V), whichallows for increased potential for antibody-dependent cell-mediatedcytotoxicity (ADCC). The IL-2 sequence is tagged with the endoplasmicreticulum retention signal, KDEL, to prevent IL-2 protein secretion fromthe endoplasmic reticulum (ER). Inclusion of the IL-2 sequence allowshaNK™ to be IL-2 independent.

EUFETS GmbH (Regensburg, Germany) conducted the transfection byelectroporation and selected multiple clones by one round of limitingdilution. A single clone from EUFETS was sent to BioReliance in order toestablish a GMP master cell bank, haNK003. Whole genome sequencing onthe selected clone confirmed that the plasmid insertion site is at asingle location on Chromosome 17 at position 15,654,977-15,661,403.

Transfection Plasmid

A plasmid was constructed by GeneArt AG based on providedspecifications. The synthetic gene pNEUKv1_FcRIL2 was assembled fromsynthetic oligonucleotides and PCR products. The fragment was clonedinto the pNEUKv1_O059 vector backbone using EcoRI and NotI restrictionsites. The pNEUKv1_O059 is a synthetic vector, containing an ampicillinresistance cassette. The promoter used for expression of the transgeneis EF-1alpha with an SV40 polyadenylation sequence. The resultingplasmid is 5,491 base pairs (bp) in length and contains human originsequences for CD16 and IL-2. Neither CD16 nor IL-2 have any transformingproperties. The plasmid DNA was purified from transformed bacteria andits concentration was determined by UV spectroscopy. The final constructwas verified by sequencing. The sequence congruence within the usedrestriction sites was 100%. The plasmid was made under TSE-freeproduction conditions.

The full nucleotide sequence of the pNEUKv1_FcRIL2 plasmid (SEQ ID NO:1)is shown here:

1 TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT TTCCCCGAAA AGTGCCACCT  61GACGTCGACG GATCGGGAGA TCTCCCGATC CCCTATGGTG CACTCTCAGT ACAATCTGCT  121CTGATGCCGC ATAGTTAAGC CAGTATCTGC TCCCTGCTTG TGTGTTGGAG GTCGCTGAGT  181AGTGCGCGAG CAAAATTTAA GCTACAACAA GGCAAGGCTT GACCGACAAT TGCATGAAGA  241ATCTGCTTAG GGTTAGGCGT TTTGCGCTGC TTCGGGATCC GCTGACCAAA AGAGCACCAA  301AGGCGCCCTG ACCTTCAGCC CCTACCTGCG CTCCGGTGCC CGTCAGTGGG CAGAGCGCAC  361ATCGCCCACA GTCCCCGAGA AGTTGGGGGG AGGGGTCGGC AATTGAACCG GTGCCTAGAG  421AAGGTGGCGC GGGGTAAACT GGGAAAGTGA TGTCGTGTAC TGGCTCCGCC TTTTTCCCGA  481GGGTGGGGGA GAACCGTATA TAAGTGCAGT AGTCGCCGTG AACGTTCTTT TTCGCAACGG  541GTTTGCCGCC AGAACACAGG TAAGTGCCGT GTGTGGTTCC CGCGGGCCTG GCCTCTTTAC  601GGGTTATGGC CCTTGCGTGC CTTGAATTAC TTCCACCTGG CTGCAGTACG TGATTCTTGA  661TCCCGAGCTT CGGGTTGGAA GTGGGTGGGA GAGTTCGAGG CCTTGCGCTT AAGGAGCCCC  721TTCGCCTCGT GCTTGAGTTG AGGCCTGGCC TGGGCGCTGG GGCCGCCGCG TGCGAATCTG  781GTGGCACCTT CGCGCCTGTC TCGCTGCTTT CGATAAGTCT CTAGCCATTT AAAATTTTTG  841ATGACCTGCT GCGACGCTTT TTTTCTGGCA AGATAGTCTT GTAAATGCGG GCCAAGATCT  901GCACACTGGT ATTTCGGTTT TTGGGGCCGC GGGCGGCGAC GGGGCCCGTG CGTCCCAGCG  961CACATGTTCG GCGAGGCGGG GCCTGCGAGC GCGGCCACCG AGAATCGGAC GGGGGTAGTC  1021TCAAGCTGGC CGGCCTGCTC TGGTGCCTGG CCTCGCGCCG CCGTGTATCG CCCCGCCCTG  1081GGCGGCAAGG CTGGCCCGGT CGGCACCAGT TGCGTGAGCG GAAAGATGGC CGCTTCCCGG  1141CCCTGCTGCA GGGAGCTCAA AATGGAGGAC GCGGCGCTCG GGAGAGCGGG CGGGTGAGTC  1201ACCCACACAA AGGAAAAGGG CCTTTCCGTC CTCAGCCGTC GCTTCATGTG ACTCCACGGA  1261GTACCGGGCG CCGTCCAGGC ACCTCGATTA GTTCTCGAGC TTTTGGAGTA CGTCGTCTTT  1321AGGTTGGGGG GAGGGGTTTT ATGCGATGGA GTTTCCCCAC ACTGAGTGGG TGGAGACTGA  1381AGTTAGGCCA GCTTGGCACT TGATGTAATT CTCCTTGGAA TTTGCCCTTT TTGAGTTTGG  1441ATCTTGGTTC ATTCTCAAGC CTCAGACAGT GGTTCAAAGT TTTTTTCTTC CATTTCAGGT  1501GTCGTGATAA TACGACTCAC TATAGGGAGA CCCAAGCTGG AATTCGCCAC CATGTGGCAG  1561CTGCTGCTGC CTACAGCTCT CCTGCTGCTG GTGTCCGCCG GCATGAGAAC CGAGGATCTG  1621CCTAAGGCCG TGGTGTTCCT GGAACCCCAG TGGTACAGAG TGCTGGAAAA GGACAGCGTG  1681ACCCTGAAGT GCCAGGGCGC CTACAGCCCC GAGGACAATA GCACCCAGTG GTTCCACAAC  1741GAGAGCCTGA TCAGCAGCCA GGCCAGCAGC TACTTCATCG ACGCCGCCAC CGTGGACGAC  1801AGCGGCGAGT ATAGATGCCA GACCAACCTG AGCACCCTGA GCGACCCCGT GCAGCTGGAA  1861GTGCACATCG GATGGCTGCT GCTGCAGGCC CCCAGATGGG TGTTCAAAGA AGAGGACCCC  1921ATCCACCTGA GATGCCACTC TTGGAAGAAC ACCGCCCTGC ACAAAGTGAC CTACCTGCAG  1981AACGGCAAGG GCAGAAAGTA CTTCCACCAC AACAGCGACT TCTACATCCC CAAGGCCACC  2041CTGAAGGACT CCGGCTCCTA CTTCTGCAGA GGCCTCGTGG GCAGCAAGAA CGTGTCCAGC  2101GAGACAGTGA ACATCACCAT CACCCAGGGC CTGGCCGTGT CTACCATCAG CAGCTTTTTC  2161CCACCCGGCT ACCAGGTGTC CTTCTGCCTC GTGATGGTGC TGCTGTTCGC CGTGGACACC  2221GGCCTGTACT TCAGCGTGAA AACAAACATC AGAAGCAGCA CCCGGGACTG GAAGGACCAC  2281AAGTTCAAGT GGCGGAAGGA CCCCCAGGAC AAGTGAAATT CCGCCCCTCT CCCCCCCCCC  2341CCTCTCCCTC CCCCCCCCCT AACGTTACTG GCCGAAGCCG CTTGGAATAA GGCCGGTGTG  2401CGTTTGTCTA TATGTTATTT TCCACCATAT TGCCGTCTTT TGGCAATGTG AGGGCCCGGA  2461AACCTGGCCC TGTCTTCTTG ACGAGCATTC CTAGGGGTCT TTCCCCTCTC GCCAAAGGAA  2521TGCAAGGTCT GTTGAATGTC GTGAAGGAAG CAGTTCCTCT GGAAGCTTCT TGAAGACAAA  2581CAACGTCTGT AGCGACCCTT TGCAGGCAGC GGAACCCCCC ACCTGGCGAC AGGTGCCTCT  2641GCGGCCAAAA GCCACGTGTA TAAGATACAC CTGCAAAGGC GGCACAACCC CAGTGCCACG  2701TTGTGAGTTG GATAGTTGTG GAAAGAGTCA AATGGCTCTC CTCAAGCGTA TTCAACAAGG  2761GGCTGAAGGA TGCCCAGAAG GTACCCCATT GTATGGGATC TGATCTGGGG CCTCGGTGCA  2821CATGCTTTAC ATGTGTTTAG TCGAGGTTAA AAAAACGTCT AGGCCCCCCG AACCACGGGG  2881ACGTGGTTTT CCTTTGAAAA ACACGATAAC CGCCACCATG TACCGGATGC AGCTGCTGAG  2941CTGTATCGCC CTGTCTCTGG CCCTCGTGAC CAACAGCGCC CCTACCAGCA GCAGCACCAA  3001GAAAACCCAG CTGCAGCTGG AACATCTGCT GCTGGACCTG CAGATGATCC TGAACGGCAT  3061CAACAACTAC AAGAACCCCA AGCTGACCCG GATGCTGACC TTCAAGTTCT ACATGCCCAA  3121GAAGGCCACC GAACTGAAAC ATCTGCAGTG CCTGGAAGAG GAACTGAAGC CCCTGGAAGA  3181AGTGCTGAAC CTGGCCCAGA GCAAGAACTT CCACCTGAGG CCCAGGGACC TGATCAGCAA  3241CATCAACGTG ATCGTGCTGG AACTGAAAGG CAGCGAGACA ACCTTCATGT GCGAGTACGC  3301CGACGAGACA GCTACCATCG TGGAATTTCT GAACCGGTGG ATCACCTTCT GCCAGAGCAT  3361CATCAGCACC CTGACCGGCT CCGAGAAGGA CGAGCTGTGA GCGGCCGCCC GCTGATCAGC  3421CTCGAACGAG ATTTCGATTC CACCGCCGCC TTCTATGAAA GGTTGGGCTT CGGAATCGTT  3481TTCCGGGACG CCGGCTGGAT GATCCTCCAG CGCGGGGATC TCATGCTGGA GTTCTTCGCC  3541CACCCCAACT TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG CATCACAAAT  3601TTCACAAATA AAGCATTTTT TTCACTGCAT TCTAGTTGTG GTTTGTCCAA ACTCATCAAT  3661GTATCTTATC ATGTCTGTGC GGTGGGCTCT ATGGCTTCTG AGGCGGAAAG AACCAGCTGG  3721GGCTCTAGGG GGTATCCCCG GATCCTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA  3781AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA  3841TCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC  3901CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC  3961CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TCACGCTGTA GGTATCTCAG  4021TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG TTCAGCCCGA  4081CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC  4141GCCACTGGCA GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG GCGGTGCTAC  4201AGAGTTCTTG AAGTGGTGGC CTAACTACGG CTACACTAGA AGAACAGTAT TTGGTATCTG  4261CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT CCGGCAAACA  4321AACCACCGCT GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC GCAGAAAAAA  4381AGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT GGAACGAAAA  4441CTCACGTTAA GGGATTTTGG TCATGAGATT ATCAAAAAGG ATCTTCACCT AGATCCTTTT  4501AAATTAAAAA TGAAGTTTTA AATCAATCTA AAGTATATAT GAGTAAACTT GGTCTGACAG  4561TTACCAATGC TTAATCAGTG AGGCACCTAT CTCAGCGATC TGTCTATTTC GTTCATCCAT  4621AGTTGCCTGA CTCCCCGTCG TGTAGATAAC TACGATACGG GAGGGCTTAC CATCTGGCCC  4681CAGTGCTGCA ATGATACCGC GAGAACCACG CTCACCGGCT CCAGATTTAT CAGCAATAAA  4741CCAGCCAGCC GGAAGGGCCG AGCGCAGAAG TGGTCCTGCA ACTTTATCCG CCTCCATCCA  4801GTCTATTAAT TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA  4861CGTTGTTGCC ATTGCTACAG GCATCGTGGT GTCACGCTCG TCGTTTGGTA TGGCTTCATT  4921CAGCTCCGGT TCCCAACGAT CAAGGCGAGT TACATGATCC CCCATGTTGT GCAAAAAAGC  4981GGTTAGCTCC TTCGGTCCTC CGATCGTTGT CAGAAGTAAG TTGGCCGCAG TGTTATCACT  5041CATGGTTATG GCAGCACTGC ATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTTC  5101TGTGACTGGT GAGTACTCAA CCAAGTCATT CTGAGAATAG TGTATGCGGC GACCGAGTTG  5161CTCTTGCCCG GCGTCAATAC GGGATAATAC CGCGCCACAT AGCAGAACTT TAAAAGTGCT  5221CATCATTGGA AAACGTTCTT CGGGGCGAAA ACTCTCAAGG ATCTTACCGC TGTTGAGATC  5281CAGTTCGATG TAACCCACTC GTGCACCCAA CTGATCTTCA GCATCTTTTA CTTTCACCAG  5341CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA AAATGCCGCA AAAAAGGGAA TAAGGGCGAC  5401ACGGAAATGT TGAATACTCA TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG  5461TTATTGTCTC ATGAGCGGAT ACATATTTGA A 

To generate the haNK003 cell line, a vial of the NK-92 (aNK) Master CellBank (MCB) (aNK COA) and 250 mg of pNEUKv1_FcRIL2 plasmid were sent toEUFETS GmbH. EUFETS thawed the MCB vial and cultured the NK-92 cells toan adequate number for transfection with the plasmid. The transfectedcells were grown in media with IL-2, X-Vivo 10, and 5% heat inactivatedHuman AB Serum for the first two days post transfection. After two days,IL-2 was no longer added to the growth media and any cells that weretransfected and producing adequate amount of IL-2 continued to grow.Multiple clones were isolated by limiting dilution and preliminarilyscreened for phenotype and Fc Receptor expression. Six (6) clones thatexhibited good viability (>70%), acceptable doubling time, expectedphenotype and positive Fc Receptor expression were sent to the GermanRed Cross GMP Testing Laboratory (GRC) for more extensive screening andfinal selection of a single clone. At GRC, all clones were tested forphenotype (including Fc Receptor expression), ADCC, cytokine profile,growth characteristics, and radiation sensitivity. The selected cellline, haNK003, was used to generate the master cell bank.

NantKwest Master Cell Bank (MCB haNK003) was manufactured from theselected cell line and tested by BioReliance. The MCB was tested forpurity, potency, identity, sterility and viral/adventitious agents. TheMCB is cryopreserved in a formulation of 10% DMSO, 40% X-Vivo 10, 50%Human AB Serum, in aliquots of 1×107 cells/vial. The total number ofvials produced from the cryopreservation for the MCB was 218.

Integration Site

DNA extract from haNK003 was provided to the CLIA/CAP certifiedNantOmics Sequencing Lab (Culver City, Calif.) for whole genomesequencing. Whole genome libraries were prepared for cell line samplesusing KAPA Hyper prep kit and sequenced on an Illumina HiSeq instrumentto provide minimum coverage of 25×, completed for haNK003. DNAsequencing data was aligned to an augmented Genome Reference ConsortiumHuman Build 37 (GRCh37, also known as hg19, originally obtained from theUniversity of California, Santa Cruz GenomeBrowser—http://genome.ucsc.edu) containing the reported plasma sequenceby bwa-mem, duplicate marked by samblaster, and indel realigned and basequality recalibrated by Genome Analysis Toolkit (GATK). Variant analysiswas performed using the NantOmics Contraster analysis pipeline todetermine variants, including single-nucleotide changes, smallinsertions or deletions (indels), copy-number changes, rearrangements,and integration sites. Integrated plasmid and resulting integrationsites were visualized by the NantOmics Genome Browser and furthercomparison and visualization was done on the UCSC Genome Browser toidentify any potential interactions with existing genomic elements.

haNK003 showed discordant read evidence of a mapping tochr17:15654977-15661403. The nearest 5′ gene TBC1D26(chr17:15,635,591-15,644,255) is 10,722 bp upstream, and the nearest 3′gene ADORA2B (chr17:15,848,231-15,871,210) is 186,828 bp downstream.Very little is known about TBC1D26, beyond being annotated as aGTPase-activating protein for Rab family protein(s) in UniProt. ADORA2Bis annotated as a membrane protein that stimulates adenylate cyclaseactivity in the presence of adenosine (Strohmeier, et al., J. Biol.Chem. 270(5):2387-2394 (1995)). No coding variants were found in the twoannotated ORFs for the coding sequence labeled pNEUKv1 FcRIL. UCSCEncode tracks and lincRNA shows evidence of a lincRNA transcriptdownstream of the insertion site (TCONS_12_00011108), however it isapproximately 2,450 bp downstream of the 3′ integration site, indicatingthis transcript is likely still intact. Investigation of a 100-waymultiple alignment of vertebrate species indicates very littlebase-level conservation across the integration site, with negative logp-values ranging from −3.874 to 1.507 with a conservation mean of 0.01and standard deviation of 0.58.

haNK003 contained no evidence of gene, transcript or regulatory breakagein the human genome integration site. Cell line haNK003's integrationwas at least 10 kbp away from any gene. The cell line is acceptable inthat there is no evidence of disruption to any known genomic features inthe target cell line human genomes.

Growth Characteristics

The growth characteristic of the clonal cell line haNK003 used togenerate the MCB haNK003 is shown in FIGS. 1A and 1B. Data was analyzedfrom the cell culture history when growing cell line haNK003 for mastercell bank cryopreservation. The mean doubling time was 65 (48-95) hoursfrom day 3 to day 29. Comparable cell densities were achieved duringpassaging demonstrating that haNK003 cells grow consistently whenpassaged every 3 to 4 days and seeded at a density of approximately0.3-0.5×10⁶ cells/mL.

Phenotype

A study was conducted to quantify the expression of a panel of sixprotein markers on the surface of haNK003 cells and to compare thehaNK003 profile to the profile of the parental cell line NK-92 (aNK).The panel of surface markers was selected to be representative ofnatural killer (NK) cells.

aNK cells express surface markers typical of an NK cell in an earlydifferentiation stage, which express a number of activation receptorsincluding NKG2D and NKp30 but lacking FcγRIIIa (CD16) and inhibitoryKIRs (Killer Immunoglobulin like Receptors). This particular surfacemarker expression profile of aNK cells gives them their unique cytotoxicproperties. Therefore, it was important to establish that the generationof the haNK003 cell line by stable transfection of a plasmid encodingthe high-affinity FcγRIIIa and intracellularly retained IL-2 (ERIL-2)did not alter the expression profile of key surface markers of theparental aNK cell line. The surface markers CD54, CD56, NKG2D, NKp30,CD3, and CD16 were analyzed and the marker expression was determined bystaining cells with specific fluorochrome-conjugated antibodies anddetecting bound antibodies by flow cytometry.

The results of the flow cytometry analysis is summarized in Table 1 andrepresentative histograms are provided in FIG. 2.

TABLE 1 Expression of surface markers. CD3 CD16 CD54 CD56 NKG2D NKp30aNK % 0.62 ± 0.06  1.02 ± 0.64 99.01 ± 0.62 98.11 ± 2.00 82.88 ± 3.1588.85 ± 7.77 haNK003 % 0.02 ± 0.11 93.15 ± 4.00 96.42 ± 3.63 97.87 ±2.60 81.95 ± 9.68 93.57 ± 2.06 % = percentage of cells positive forexpression ± standard deviation

haNK003 and aNK express comparable amounts of CD54, CD56, NKG2D andNKp30 as determined by median fluorescent intensity. In addition, thepercentage of cells expressing these markers is equivalent. NeitherhaNK003 nor aNK express CD3 which is a T cell marker. As expected,haNK003 expresses the CD16 marker while aNK does not.

The generation of the haNK003 cell line has not altered the expressionof key surface markers of the parental aNK cell line and has only addedadditional functionality in the form of the expression of CD16.

Cytotoxicity

Natural cytotoxicity of haNK003 cell line was evaluated against celllines K562, Raji, SKOV3, and SKBR3 at various effector to target ratios(E:T). ADCC activity of haNK003 was also evaluated against cell linesRaji, SKOV3, and SKBR3 at various E:T ratios. The sensitivity ofdifferent target cell lines to haNK killing by natural cytotoxicityvaries, with the K562 cell line being the most sensitive and the solidtumor cell lines (SKOV3 and SKBR3) being less sensitive (FIGS. 3A, 3B,3C and 3D). Some variation in haNK003 ADCC activity toward differenttarget cell lines was also observed, with the highest specific lysisobserved in Raji cells in combination with Rituximab (FIGS. 4A, 4B and4C).

The results demonstrate that haNK003 cells exhibit natural cytotoxicityin the presence of several cancer cells, and are capable of enhancedspecific lysis via ADCC with antibodies. The specifications and resultsfor haNK™ MCB (haNK003) are provided in Table 2.

TABLE 2 Specifications of haNK003 cells. Test Description TestSpecification haNK003 Results Avg. Cell suspension ≥0.9 mL 1.07 mLvolume/vial Avg. Viability ≥85% 89% (post-bank thaw) Avg. Viability ≥85%97% (post-bank passaged) Avg. Total Viable Report result 7.13 × 10⁶Cells/vial Natural Killer (NK) Report result - target greater 5:1 E:TRatio with Herceptin cell assay & tumor than 50% at 20:1 E:T ratio (3.3μg/mL) vs SKOV3 71.1%; target cell - ADCC assay 1:1 E:T Ratio withRituxan with Herceptin and Rituxan (3.3 μg/mL) vs D0HH2 52.8% Identityby Fluorescent ≥90% CD56+ 98.0% Monoclonal Antibody Staining ≥90% CD16+92.7% for Surface Antigens ≤5% CD3+ 0.02% CO1 Barcode Assay for CellCell line is of human Origin Pass Line Identification Isolator SterilityTesting No bacterial or fungal Growth Pass Using a Direct InoculationMethod Test for Presence of Agar- No mycoplasma Detected Pass Cultivableand Non-agar Cultivable Mycoplasma In Vitro Assay for the Negative forthe presence of viral Performed on aNK MCB Presence of Bovine Virusescontaminants (haNK003 starting material) According to 9 CFR RequirementsNegative 28-day In Vitro Assay for the Negative for the presence ofviral Pass Presence of Viral Contaminants contaminants Test for thePresence of Negative for the presence of Pass Inapparent Virusesadventitious viral contaminants Transmission Electron Report Result Noextraneous agents observed in Microscopic Examination of Cell the 200cell profiles examined Cultures (200 Cell profiles) Quantitative ProductEnhanced Report Result The concentration of RT units in ReverseTranscriptase (Q-PERT) the sample is <5.00 × 10⁻⁷ Assay for theDetection of units/mL Retrovirus in Biological Samples Detection of 14Viruses by Real Report Result All virus negative except EBV_(a) TimePolymerase Chain Reaction (detection limit = 10 copies) Assays (HumanPanel I) Assessment of Production of Does not actively produce EBVPerformed on aNK MCB Infectious Epstein Barr Virus (haNK003 startingmaterial) (EBV) by NK-92 Cells NK-92 cells do not pose a risk for EBVinfection^(a) ^(a)Although EBV virus genome is detected, tests withNK-92 (aNK) determined that cells do not cause infection of EBV.Infectivity studies were conducted by co-culturing aNK cells (irradiatedand non-irradiated) with B-lymphocytes to determine if the aNK cellsrelease viral particles capable of infecting normal cells. Resultsshowed no indication of proliferation or outgrowth of B-lymphocytes,indicating that aNK cells do not pose a risk for EBV infection.

Example 2. Effect of Irradiation on In Vitro Proliferative Capacity andFunctionality

haNK™ cells are irradiated to mitigate the risk of uninhibitedproliferation. The effects of irradiation on in vitro proliferativecapacity and functionality were evaluated. These studies demonstratethat irradiation at 10 Gy inhibits the proliferative capacity of atleast 99.9% of haNK cells while still maintaining functional activityfor at least 6 hours post-irradiation.

haNK003 cells exhibit both natural (direct) cytotoxicity andantibody-dependent cell-mediated cytotoxicity (ADCC). In both cases,target cell antigens are recognized by activating receptors on NK cells.For natural cytotoxicity, these target cell antigens are stress antigenscharacteristic of virally infected or transformed cells. For ADCC, theantigens are tumor specific antigens that are recognized by an antibody,which in turn binds to the NK cell activating receptor, FcγRIIIa (CD16),through its constant (Fc) region.

The interaction of the NK cell with target cell ligands (either throughdirect interaction or through an antibody-mediated interaction) resultsin the formation of a cellular junction and subsequent release ofperforins and granzymes. This in turn induces an apoptotic processwithin the target cell leading to disintegration of the cell membraneand target cell death.

A study was conducted to determine the level of natural cytotoxicity andADCC activity and the duration of that activity following gammairradiation at 10 Gy on pre-formulated cells. For this study, 1.5×107cells per flask were irradiated. Although the number of cells subjectedto irradiation was not equivalent to that which will be used inmanufacture of a clinical dose, assay of the functionality followingirradiation will provide insight for manufacturing capabilities.

For the purposes of these experiments, haNK003 cells were irradiated at10 Gy using an X-ray irradiator. Non-irradiated cells were subjected tothe same manipulations without irradiation. Target cells were selectedto represent sensitivities to different mechanisms of killing by NKcells. For example, K562 cells are highly sensitive to killing bynatural cytotoxicity while DOHH is partially sensitive to killingthrough natural cytotoxicity, but sensitive to ADCC with the appropriateantibodies. Irradiated and non-irradiated cells were assayedside-by-side for specific cytotoxicity using a flow cytometry basedassay that was developed in-house. Target cells were labeled with agreen fluorescent dye with long aliphatic tails (PKH67) to stablyincorporate the dye into lipid regions of the cell membrane. Cell lysiswas monitored by propidium iodide staining.

Analysis of natural cytotoxic activity demonstrates that the impact ofirradiation varies with the target cell and time post-irradiation. Forsensitive cells such as K562, natural cytotoxic activity was maintainedwithin 6 hours of irradiation but was reduced by 40% or more after 24hours across all effector to target ratios (E:T) (FIG. 5). For DOHH2,natural cytotoxic activity was also retained at 6 hours post-irradiationbut was reduced by 14% or more after 24 hours across all effector totarget ratios and reduced by as much as 50% at a E:T ratio of 10:1 (FIG.6).

The level of rituximab-mediated ADCC activity with DOHH2 targets wasalso maintained by irradiated cells at 6 hours. However,rituximab-mediated ADCC activity for irradiated cells at 24 hours wasreduced by about 16% or more from the activity seen with cells at 6hours, and non-irradiated cells at 24 hours (FIG. 7). As expected,Herceptin (trastuzumab) did not induce any ADCC killing of DOHH2 targetcells in combination with haNK003 (FIG. 4), nor did the antibodies alone(rituximab or trastuzumab) induce any DOHH2 target cell killing (datanot shown).

Relevant levels of cytotoxic activity and ADCC activity are maintainedfor at least 6 hours after irradiation of pre-formulated haNK003 cells.

Example 3. Characterization of IL-2 Release

A study was conducted to analyze the amount of IL-2 released by haNK003cells into the culture medium as well as the amount ofintracellular-retained IL-2 in haNK003 cells at various time points.Amounts of IL-2 were measured in supernatants to determine IL-2 releaseby haNK003. Amounts of IL-2 were measured in cell pellet lysates todetermine the total levels of intracellular IL-2 in haNK003. Sampleswere analyzed pre- and post-irradiation to determine the impact ofirradiation on IL-2 release and intracellular IL-2 levels.

Two separate assays were performed (Run #1 and Run #2) where haNK003cells were cultured in T-75 flasks and irradiated at 0 Gy (noirradiation) or 10 Gy (irradiation) using an X-ray irradiator. haNK003cells (non-irradiated and irradiated) were then cultured in X-Vivo 10with 5% heat-inactivated Human AB Serum for up to 48 hours. Samples fromculture supernatants as well as cell pellets were collected for analysisat various time points. Cell pellets were lysed using a detergent-basedsolution to quantify total intracellular IL-2. The IL-2 concentration inculture supernatant and in cell lysate samples was measuredindependently at NantKwest or by AllCells, LLC using two differentdetection methods (sandwich ELISA or multiplex ELISA).

The IL-2 concentration values measured by the two different methodsdiffer by a factor of five on average for the same samples. Both methodsshow a linear increase of IL-2 release over time for both irradiated andnon-irradiated cells (Table 3 and FIGS. 8A, 8B, 8C, and 8D). IL-2concentration in the culture supernatants of irradiated cells tends tobe higher than non-irradiated cells at all time points, although thedifference varied between assay runs #1 and #2.

TABLE 3 IL-2 Released (pg/mL) per 1 × 10⁶ Cells of Irradiated vs.Non-irradiated haNK003 at Different Time Points (average and standarddeviation [StDev] for duplicate reads) Sandwich ELISA Multiplex ELISAI1-2 Run#1 Run#2 Run#1 Run#2 (pg/mL) Time Average StDev Average StDevAverage StDev Average StDev No  6 h 68.59 5.52 78.62 6.00 290.06 11.89666.99 51.20 Irradiation 12 h 139.67 9.93 117.29 1.92 458.38 4.48 851.7418.34 24 h 217.73 6.68 222.64 9.05 734.75 24.11 1578.50 8.80 48 h 575.6628.84 544.32 9.88 1548.19 33.72 2944.94 207.54 Irradiation  6 h 194.6711.21 129.44 0.62 689.11 12.69 961.78 3.70 10 Gy 12 h 299.49 5.42 181.142.38 828.70 70.00 1254.33 8.17 24 h 407.17 20.92 305.67 13.46 1277.7844.17 1804.96 59.97 48 h 760.03 52.77 575.04 8.03 2706.81 110.53 3116.8567.10 h—hours

The total intracellular IL-2 concentration values measured by the twodifferent methods differ by a factor of five on average for the samesamples (Table 4 and FIGS. 9A, 9B, 9C and 9D). Both methods show anincrease of total intracellular IL-2 in non-irradiated cell lysates overtime while the amount of total intracellular IL-2 in irradiated celllysates decreases over time. At 6 hours in culture, levels of totalintracellular IL-2 were comparable between irradiated and non-irradiatedcells as measured by both methods. In irradiated cells, these levelsdecrease after 48 hours in culture.

TABLE 4 Total Intracellular IL-2 Content (pg) per 1 × 10⁶ Cells ofIrradiated vs. Non-irradiated haNK003 at Different Time Points (averageand standard deviation [StDev] for duplicate reads) Sandwich ELISAMultiplex ELISA Run#1 Run#2 Run#1 Run#2 I1-2 (pg) Time Average StDevAverage StDev Average StDev Average StDev No  6 h 1874.67 257.77 2147.01545.61 10579.41 256.64 12918.29 617.75 Irradiation 12 h 1754.12 181.892129.07 161.22 9255.57 377.00 11974.20 179.42 24 h 2550.76 101.923014.07 16.52 14267.76 5.22 16275.56 321.19 48 h 3350.60 277.37 4788.3950.06 15273.12 356.81 24701.64 103.56 Irradiation  6 h 1851.47 208.931932.82 130.67 11614.02 3653.73 11588.93 1244.94 10 Gy 12 h 1297.4156.05 1678.55 41.01 8007.80 429.63 10186.55 697.17 24 h 1053.23 140.841374.36 48.56 6902.43 2143.72 9241.67 75.20 48 h 666.46 54.55 930.6744.93 4394.21 223.74 6985.23 36.66 h—hours

To simulate the release of IL-2 upon cell necrosis, cells were subjectedto hypotonic shock. This level was compared to the total intracellularIL-2 concentration measured in cell lysates prepared using adetergent-based method. The solubilized IL-2 concentration valuesmeasured by the two methods (sandwich or multiplex ELISA) differ by afactor ˜10 for the same samples (Table 5 and FIGS. 10A and 10B). IL-2concentration in lysates from hypotonic shock was between 181.93-289.54pg/106 cells (sandwich ELISA) and 2619.15-3301.02 pg/106 cells(multiplex ELISA), which represents on average between 14% (sandwichELISA) and 27% (multiplex ELISA) of the total intracellular IL-2concentration measured in cell lysates prepared using a detergent-basedlysis.

TABLE 5 Amount of Solubilized IL-2 (pg) per 1 × 10₆ Cells of haNK003Cells (average and standard deviation [StDev] for duplicate reads).Sandwich ELISA Multiplex ELISA I1-2 (pg) Lysis Run#1 Run#2 Run#1 Run#2Sample Solution Average StDeV Average StDeV Average StDev Average StDevhaNK003 Detergent 1604.17 65.86 1685.23 49.92 10095.68 1451.30 11636.49311.54 (Triton 0.1%) Hypotonic 181.93 21.80 289.54 40.03 2619.15 186.893301.02 14.85 (H2O) Controls Detergent 0.43 0.61 0.92 1.30 0.00 0.000.00 0.00 (Triton 0.1%)

Multiplex ELISA IL-2 quantification values were between 5 and 10 timeshigher than data from sandwich ELISA method. Although there isvariability in the absolute values from these two methods, the trendsare consistent between both data sets and are summarized below. Thisdata will be useful to allow NantKwest to continue characterizing IL-2secretion and intracellular IL-2 levels from haNK003 cells as theproduct is further developed.

In summary, haNK003 cells release a detectable amount of IL-2 into theculture medium (10 to 40 pg/hour per million cells), and the amount ofIL-2 released by live cells under steady state culture conditionsrepresents on average less than 10% of the total intracellular IL-2stock.

Irradiation of the haNK003 cells with a dose of 10 Gy increases theamount of released IL-2 over a period of 48 hours, likely reflecting thepresence of dying cells. Furthermore, irradiation does not result inrelease of IL-2 all at once, but gradually, over time.

To simulate release of IL-2 upon necrotic cell death, haNK003 cells weresubjected to hypotonic shock. The amount of IL-2 released under theseconditions represents between 14 and 27% of the total intracellular IL-2determined in Triton X-100 lysates (which solubilizes proteins from allintracellular compartments).

Overall, haNK003 cells secrete low levels of IL-2 (493.8 pg/mL forirradiated cells and 276.1 pg/mL for non-irradiated cells over 6 hourswhen averaged across all runs from both methods). Taken together, thelow level of IL-2 secreted by haNK003 cells, the extremely shorthalf-life of IL-2 in the plasma, and the lack of persistence ofirradiated haNK003 cells in vivo, suggest that IL-2 release by infusedhaNK003 is unlikely to cause a clinical adverse effect.

The effects of irradiation on in vitro proliferative capacity andfunctionality, as tested in development studies with pre-formulatedcells, demonstrate that haNK003 cells have limited proliferation (lessthan 0.1% of cells) in vitro and that levels of cytotoxic activity andADCC activity are maintained for at least 6 hours after irradiation.

IL-2 secretion and intracellular IL-2 levels of haNK003 cells pre- andpost-irradiation demonstrate that haNK003 cells secrete low levels ofIL-2. haNK003 cells, irradiated or non-irradiated, do not releaseamounts of IL-2 that would be anticipated to have an adverse effect inhumans.

Example 4. Tolerability and Tumorigenicity of Single-Dose haNK003 CellsAdministered Intravenously

Natural killer (NK) cells are potent cytotoxic effector cells for cancertherapy and potentially for viral infections. NantKwest has successfullyestablished unique NK cell-based platforms to produce GMP-gradeactivated NK (aNK cells). aNK cells are actively being pursued in theclinic for cellular therapy of patients with a variety of advancedhematological malignancies and solid tumors. Recently a GMP-gradeplasmid-transfected variant of NK-92 expressing the high-affinity CD16receptor was developed utilizing a novel transfection vector containingthe ER IL-2 gene, enabling the resulting haNK003 cells to growindependently of IL-2. Expression of the high-affinity CD16 receptorenables haNK003 cells display high antibody-dependent cell-mediatedcytotoxity (ADCC) in combination with rituximab and trastuzumab anddaratumumab against target cell lines that were not killed by theparental NK-92 cells. One cell clone was selected to generate the MasterCell Bank, haNK003, which is in clinical development.

Materials and Methods

Eighteen (18) NOD.CB17-Prkdc^(scid)/J (NOD/SCID) mice (9 male and 9female) were used to investigate tolerability and tumorigenicity ofsingle-dose haNK003 cells administered intravenously in NOD/SCID mice.The mice were obtained from Jackson Laboratory (610 Main Street BarHarbor, Me. 04609 US).

18 NOD/SCID mice were selected and randomly assigned to 3 groups with 6mice (3 male and 3 female) per group based on animal body weight. Micewere intravenously administered with a single dose of PBS,non-irradiated or 10-Gy irradiated haNK003 cells, as indicated in Table6. Animals were then monitored by daily observation and twice weeklyanimal body weight measurement. After 5 weeks, animals were euthanizedand major organs were harvested and processed for furtherhistopathological examination and immunohistochemistry analysis withanti-CD56 antibody.

TABLE 6 Study Design Animal Animal Dosing Group No. Sex Treatment DoseSchedule Route A 3 M PBS / Single IV Dose 3 F PBS / Single IV Dose B 3 MNon- 1 × 10⁷ cells Single IV irradiated Dose haNK003 3 F Non- 1 × 10⁷cells Single IV irradiated Dose haNK003 C 3 M Irradiated 1 × 10⁷ cellsSingle IV haNK003 Dose 3 F Irradiated 1 × 10⁷ cells Single IV haNK003Dose

For cell culture, the haNK003 cells were cultured in X-Vivo10 medium(Cat # BE02-055Q) supplemented with 5% heat inactivated human AB serum(Cat # IPLA-SERAB-HI, Innovative Research), 100 U penicillin/ml and 100μg/ml streptomycin (Corning, Cat #30-002-CI).

For irradiation, the haNK003 cells growing in an exponential growthphase were harvested and counted for viable cell number and viability.On the appropriate day, half of the haNK003 cells were irradiated with adose of 1000 cGy using JL Shephard Mark 1 Model 68 ₁₃₇Cs irradiator(service provided by the Department of Radiation Oncology, theUniversity of California, Irvine, Calif. 92697).

For cell preparation for dosing, non-irradiated or irradiated haNK003cells were kept on ice during transportation to the animal facility(1124 W. Carson Street, Torrance, Calif., 90502). Cells were washedtwice with cold PBS, and then re-suspended in an appropriate amount ofcold PBS and passed through a 40 μm cell strainer to make a single cellpreparation with a final cell density of 5×10₇ viable cells/ml. Cellviability was determined with the Vi-CELL cell viability analyzer, andonly cells with over 85% viability were used for this study. Then thesenon-irradiated or irradiated haNK003 cells were stored at roomtemperature for IV dosing animals in an appropriate Group, respectively.

18 NOD/SCID mice were selected and randomly assigned to 3 groups with 6mice (3 male and 3 female) per group based on animal body weight.

On an appropriate day, each animal in Group A received a specific amountof PBS respectively. The dosing volume was 200 μl, regardless ofindividual animal body weight. The dosing route was IV injection viatail vein, and the dosing schedule was a single dose, as indicated inthe study protocol. On an appropriate day, each animal in Groups B and Creceived 1×10⁷ non-irradiated and irradiated haNK003 cells in 200 μlPBS, respectively. The dosing volume was 200 μl, the dosing route was IVinjection via tail vein; and the dosing schedule was a single dose.

Animals were observed once daily for general appearance. Clinicalobservations were conducted twice daily and recorded. Animals wereroutinely monitored after treatment for effects on normal behavior suchas mobility, food and water consumption (by visual estimation), and bodyweight (gain/loss).

Summary statistics, including mean and standard error of the mean (SEM),were provided for the animal body weight of each group at each timepoint. Statistical analyses of difference in animal body weight changeamong the groups were evaluated using two-way ANOVA with repeatedmeasures followed by Bonferroni test. All the data were analyzed usingGraphPad Prism software version 5. p<0.05 was considered to bestatistically significant.

Results

Non-irradiated or irradiated haNK003 cells at the dose of 1×10⁷ cells,administered intravenously as a single agent, were well-tolerated with amaximum average body weight loss of 5.2% and 4.4%, respectively. Therewas no significant body weight loss in NOD/SCID mice when administeredwith a single dose of either irradiated or non-irradiated haNK003 cells(1×10⁷), compared to PBS-treated control group, as indicated in Table 7,FIG. 11. There was not any treatment related mortality that occurredover 5-weeks of observation in all treatment groups, as shown in Table7.

There were not any visible tumor masses in all the tissues and organsassessed, including brain, heart, liver, lung, kidney, spleen and thymusin all treatment groups, regardless of male or female mice. Results fromhistology analysis and IHC staining using anti-CD56 antibody confirmedthat there were not any haNK003 cell-related lymphoid aggregates intissues and organs, including brain, bone marrow, heart, liver, lung,kidney, spleen and thymus, suggesting that both non-irradiated andirradiated haNK003 cells have no tumorigenic potential in NOD/SCID mice.

Pathological examination of all the specimens obtained in the study,including brain, bone marrow, heart, liver, lung, kidney, spleen andthymus, indicated that there was not any significant haNK003 treatmentrelated toxicities in either non-irradiated or irradiated haNK003cells-treated group, compared to PBS treated group.

TABLE 7 Effect of haNK003 administered intravenously on animal bodyweight, mortality and tumorigenicity in NOD/SCID mice. Dosing MWL^(a)Mortality^(c) Tumori- Group Treatment Dose Schedule (%) P value^(b)(n/total) genicity A PBS / Single / / 0/6 NO Dose B Non-irradiated 1 ×10⁷ cells Single 5.2 P > 0.05 0/6 NO haNK003 Dose C Irradiated 1 × 10⁷cells Single 4.4 p > 0.05 0/6 NO haNK003 Dose Note: ^(a)MWL: maximumbody weight loss; ^(b)p-value (two-way ANOVA with repeated measuresfollowed by Bonferroni test) vs. PBS treatment; ^(c)(n/total): number oftreatment related animal deaths per total number of animals in anindividual group.

Either Irradiated or non-irradiated haNK003 cells as a single agentadministered intravenously at the dose of 1×10⁷ cells werewell-tolerated in both male and female NOD/SCID mice. There was nosignificant body weight loss associated with either irradiated ornon-irradiated haNK003 treatment. There was not any treatment relatedmortality that occurred over 5-weeks of observation in all treatmentgroups. There were not any significant pathological changes in the majororgans including brain, bone marrow, heart, liver, lung, kidney, spleenand thymus. Most importantly, there was no tumorigenic potential ofirradiated or non-irradiated haNK003 cells in both male and femaleNOD/SCID mice.

Example 5. Tolerability and Tumorigenicity of Repeated-Dose haNK003Cells Administered Intravenously Materials and Methods

Eighteen (18) NOD.CB17-Prkdcscid/J (NOD/SCID) mice (9 male and 9 female)were used to investigate tolerability and tumorigenicity of single-dosehaNK003 cells administered intravenously in NOD/SCID mice. The mice wereobtained from Jackson Laboratory (610 Main Street Bar Harbor, Me. 04609US).

18 NOD/SCID mice were selected and randomly assigned to 3 groups with 6mice (3 male and 3 female) per group based on animal body weight. Micewere intravenously administered with repeated dose of PBS,non-irradiated or 10-Gy irradiated haNK003 cells once weekly for 4weeks, respectively as indicated in Table 8. Animals were then monitoredby daily observation and twice weekly animal body weight measurement.After 5 weeks, animals were euthanized and major organs were harvestedand processed for further histopathological examination andimmunohistochemistry (IHC) analysis with anti-CD56 antibody.

TABLE 8 Study Design. Animal Animal Dosing Group No. Sex Treatment DoseSchedule Route A 3 M PBS / QW × 4 IV 3 F PBS / QW × 4 IV B 3 M Non- 1 ×10⁷ cells QW × 4 IV irradiated haNK003 3 F Non- 1 × 10⁷ cells QW × 4 IVirradiated haNK003 C 3 M Irradiated 1 × 10⁷ cells QW × 4 IV haNK003 3 FIrradiated 1 × 10⁷ cells QW × 4 IV haNK003

For cell culture, haNK003 cells were cultured in X-Vivo10 medium (Cat #BE02-055Q) supplemented with 5% heat inactivated human AB serum (Cat #IPLA-SERAB-HI, Innovative Research), 100 U penicillin/ml and 100 μg/mlstreptomycin (Corning, Cat #30-002-CI).

For irradiation, haNK003 cells growing in an exponential growth phasewere harvested and counted for viable cell number and viability. On theappropriate day, half of the haNK003 cells were irradiated with a doseof 1000 cGy using JL Shephard Mark 1 Model 68 ₁₃₇Cs irradiator (serviceprovided by the Department of Radiation Oncology, the University ofCalifornia, Irvine, Calif. 92697).

For cell preparation for dosing, Non-irradiated or Irradiated haNK003cells were kept on ice during the transportation to animal facility(1124 W. Carson Street, Torrance, Calif., 90502). Cells were washedtwice with cold PBS, and then re-suspended in appropriate amount of coldPBS and passed through 40 μm cell strainer to make single cellpreparation with a final cell density of 5×10₇ cells/ml. Cell viabilitywas determined with Vi-CELL cell viability analyzer, and only cells withover 85% viability were used for this study. Then these non-irradiatedor irradiated haNK003 cells were stored at RT for IV dosing animals inan appropriate group, respectively.

18 NOD/SCID mice were selected and randomly assigned to 3 groups with 6mice (3 male and 3 female) per group based on animal body weight.

On an appropriate day, each animal in Group A received 200 μl of PBS,regardless of an individual animal body weight. The dosing route was IVinjection via tail vein, and the dosing schedule was once weekly fortotal 4 weeks, as indicated in the study protocol (Appendix 1). Eachanimal in Groups B and C received 1×10₇ non-irradiated and irradiatedhaNK003 cells in 200 μl PBS, respectively. The dosing volume was 200 μl,the dosing route was IV injection via tail vein, and the dosing schedulewas once weekly for total 4 weeks, as indicated in the study protocol.

Animals were observed once daily for general appearance. Clinicalobservations were conducted twice daily and recorded. Animals wereroutinely monitored after treatment for effects on normal behavior suchas mobility, food and water consumption (by visual estimation), and bodyweight (gain/loss).

Summary statistics, including mean and standard error of the mean (SEM),were provided for the animal body weight of each group at each timepoint. Statistical analyses of difference in animal body weight changeamong the groups were evaluated using two-way ANOVA with repeatedmeasures followed by Bonferroni test. All the data were analyzed usingGraphPad Prism software version 5. p<0.05 was considered to bestatistically significant.

Results

Non-irradiated or irradiated haNK003 cells as a single agentadministered intravenously once weekly for 4 weeks, were well-toleratedwith a maximum average body weight loss of 3.4% and 4.9%, respectively.Regardless male or female mice, there was no significant body weightloss in NOD/SCID mice when administered with either non-irradiated orirradiated haNK003 cells (1×10⁷), compared to PBS-treated control group,as indicated in Table 9, FIG. 12. There was not any treatment relatedmortality that occurred over 5-week of observation in all treatmentgroups, as summarized in Table 9.

TABLE 9 Effect of haNK003 cells on animal body weight, mortality andtumorigenicity in NOD/SCID mice. Dosing MWL^(a) Mortality^(c) Tumori-Group Treatment Dose Schedule (%) P value^(b) (n/total) genicity A PBS /QW × 4 1.0 / 0/6 NO B Non-irradiated 1 × 10⁷ cells QW × 4 3.4 P > 0.050/6 NO haNK003 C Irradiated 1 × 10⁷ cells QW × 4 4.9 p > 0.05 0/6 NOhaNK003 Note: ^(a)MWL: maximum body weight loss; ^(b)p-value (two-wayANOVA with repeated measures followed by Bonferroni test) vs. PBStreatment; ^(c)(n/total): number of treatment related animal deaths pertotal number of animals in an individual group.

Macropathological and histopathological examination of all the specimensobtained in this study, including brain, bone marrow, heart, liver,lung, kidney, spleen and thymus, indicated that there was not anysignificant haNK003 treatment related toxicities in these organs ineither nonirradiated or irradiated haNK003 cell treated groups, comparedto PBS-treated control group. There was no splenomegaly identified inall animals. There were no tumor masses found by gross findings in alltreatment groups. Results from IHC using anti-CD56 antibody confirmedthat there were not any haNK003 cell-related lymphoid aggregates intissues and organs including bone marrow, brain, liver, lung, heart,kidney, spleen, thymus, etc, over 5-week of follow-up, suggesting thatthere was no irradiated or non-irradiated haNK003 cells related leukemiaor tumor development in NOD/SCID mice over these 5 weeks.

There was no edema, no degeneration, and no necrosis present in allthese tissues. Compared to PBS control group, very focal mild andminimum fatty changes in the liver tissues were noted in non-irradiatedand irradiated haNK003 treatment groups, respectively. It is alsonoteworthy that the liver parenchyma in PBS, non-irradiated orirradiated haNK003 treatment groups showed rare minute foci of mixedinflammatory cells including neutrophils and mononuclear cells, sincethese small cluster cells were CD56 negative, suggesting these mixedinflammatory cells very likely resulted from repeated procedures (tailvein injection), not related with haNK003 treatment.

In summary, non-irradiated and irradiated HaNK003 cells werewell-tolerated. There were not any significant haNK003 treatment relatedtoxicities or tumorigenicity issues in NOD/SCID mice at this dosingregimen.

Example 6. Comparison of aNK and haNK003 Natural Cytotoxic Activity

A major mechanism by which Natural Killer (NK) cells kill target cellsis through the formation of a cellular junction and the subsequentsecretion of perforin and granzymes. This in turn induces an apoptoticprocess within the target cell leading to disintegration of the plasmamembrane and cell death. Target cells can be recognized by theexpression of stress antigens characteristic of virally infected cellsand/or transformed cells. The recognition and subsequent killing of atarget cell through engagement of stress antigens with activatingreceptors on NK cells is termed natural (or direct) cytotoxicity.

As natural cytotoxicity is a major functional characteristic of NKcells, an analysis of this functionality in cell variants and comparisonto activated NK-92 cell (aNK) activity can help establish the impact ofa particular genetic modification of aNK cells.

Materials and Methods

Six (6) cell lines representative of liquid and solid tumors wereselected as targets. Targets were also selected so as to represent arange of sensitivities to aNK cell killing, with SR-91 being relativelyinsensitive to killing and K562 being highly sensitive to killing, andothers falling somewhere in between. Targets and effector cells (haNK003or aNK) were co-incubated for 4 hours at 37° C. and target cell killingwas determined by flow cytometry using an in-house developed method todetermine the specific cytotoxicity of effector cells against targetcells loaded with PKH67 fluorescent dye staining. The PKH67 FluorescentCell Linker Kits use proprietary membrane labelling technology(Sigma-Aldrich) to stably incorporate a green fluorescent dye with longaliphatic tails (PKH67) into lipid regions of the cell membrane. Due toits longer aliphatic carbon tails, PKH67 exhibits reduced cell-celltransfer. PKH67 is well suited for cytotoxicity assays that usepropidium iodide as viability probe. Staining with propidium iodidedifferentiates dead target cells (which will be doubly stained) fromdead effector cells (aNK or haNK cells).

For cell culture, aNK cells were cultured in X-Vivo 10 mediumsupplemented with 5% heat inactivated human AB serum (from CMV-negativetested donors) and 500 IU/ml recombinant human IL-2. aNK cultures werepassaged every 1-4 days in order to keep the cell density >10e5 cells/mLand <10e6 cells/mL. haNK003 cells were cultured in X-Vivo 10 mediumsupplemented with 5% heat inactivated human AB serum (from CMV-negativetested donors), without IL-2. haNK003 cultures were passaged every 1-4days in order to keep the cell density >10e5 cells/mL and <10e6cells/mL. K562, Daudi, DOHH2, HL-60, SR-91, and SKOV3 cells werecultured in RPMI-1640 supplemented with 10% heat inactivated fetalbovine serum (FBS) and a cocktail of antibiotics/antimycotic. Cellsgrowing in suspension were passaged by simple dilution, while adherentcells (SKOV3) were passaged by trypsinization of the culture usingTrypLE™. Passages were every 2-5 days (depending of the particulardoubling time of the cell line), or whenever the culture medium appearedyellow (acidic) indicating spent medium.

For sample preparation, suspension-growing cell lines were resuspendedby up and down pipetting of the cell cultures. Adherent target celllines (SKOV3) were enzymatically detached from culture vessels usingTrypLE™ and resuspended by up and down pipetting of the trypsinizedcells pellet. Cells viability was determined by manual counting (trypanblue exclusion method). Dilutions of target and effector cells to therequired cell concentrations were made in RPMI-1640 supplemented with10% heat-inactivated FBS and antibiotics/antimycotic. Effector andtarget cells were mixed at different effector to target (E:T of 20:1,10:1, 5:1, 2.5:1, 1.25:1, 0.62:1, 0.31:1, and 0.15:1) ratios in a96-well plate and co-incubated for 4 hours in a 5% CO₂ atmosphere 37° C.incubator.

Samples were analyzed on a MACSQuant flow cytometer (Miltenyi), usingthe B1 (FITC) and B3 (PerCP-Vio700/PI) fluorescence channels. Targetsalone −PI and +PI were used to determine the B1/B3 compensationparameters.

Cytotoxicity % was calculated by the formula=[(% FITC+/PI+ cells insamples)−(% FITC+/PI+ in Targets +PI only)]/[100−(% FITC+/PI+ inTargets+PI only)]

Results

aNK and haNK003 cells used for this study were thawed on Jul. 22, 2016.All the target cell cultures used in this study were less than 8 weeksold. Target cells and effector cells cultures were passaged at most 48hours before the assay.

The results shown in FIGS. 13-18 confirm the sensitivity of the targetcell lines to killing by aNK with K562 being the most sensitive (75%specific lysis at low effector to target ratios of 1:1). Daudi, DOHH2and HL-60 demonstrated intermediate sensitivity requiring a highereffector to target ratio (10:1) to achieve 65-80% specific lysis. SKOV3and SR-91 were the most resistant requiring effector to target ratios inexcess of 10:1 to achieve specific lysis of approximately 40%. In eachcase, the natural cytotoxic activity of haNK003 was comparable to thatof aNK and generally followed the same activity profile within the errorranges of the experiment.

aNK and haNK003 cells show comparable cytotoxic activity against the sixcancer cell lines tested demonstrating that the natural cytotoxicactivity of aNK cells is essentially the same despite the geneticmodification used to create the haNK003 cells. haNK003 cells and aNKcells are comparable in functionality with respect to natural cytotoxicactivity.

Example 7. Evaluation of the Anti-Tumor Activity of haNK003 inMDA-MB-453 Human Breast Carcinoma Subcutaneous Mouse Model

In this study, the anti-tumor activity of haNK003 cells as a singleagent were evaluated in the MDA-MB-453 human breast carcinomasubcutaneous xenograft model in female NOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ (NOD scid gamma, NSG) mice.

Materials and Methods

Twelve NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ (NOD scid gamma, NSG) micewere used to evaluate the anti-tumor activity of haNK003 in theMDA-MB-453 human breast carcinoma subcutaneous (s.c.) xenograft model.The mice were obtained from Jackson Laboratory (610 Main Street BarHarbor, Me. 04609 US).

MDA-MB-453 HER2− positive human breast carcinoma s.c. xenograft modelwas established in female NSG mice. Once the average size of tumorsreached about 100 mm³, treatment was initiated, and anti-tumor activityof haNK003 cells as a single agent were evaluated in this xenograftmodel. Other test articles were evaluated in parallel under the protocolLABC-X01612, but only the results of haNK003 in comparison to PBS arepresented herein. The haNK003 treatment groups and dosing regimen designare described in Table 10.

TABLE 10 Study Design. Animal Test Article Dose Dosing Dosing GroupTreatment No. Dose Concentration Volume Schedule Route A PBS 4 / / 200μl Twice weekly × 4 IV F Irradiated 4 2.5 × 10⁶ cells 1.25 × 10⁷cells/ml 200 μl Twice weekly × 4 IV haNK003 G Irradiated 4  1 × 10⁷cells   5 × 10⁷ cells/ml 200 μl Twice weekly × 4 IV haNK003

For tumor cell culture, MDA-MB-453 human breast carcinoma cells (ATCC,Cat # HTB-131) were cultured in ATCC-formulated Leibovitz's L-15 medium(ATCC, Cat #30-2008) supplemented with 10% heat-inactivated FBS(GeneTex, Cat # GTX73252), 100 U penicillin/ml and 100 μg/mlstreptomycin (Corning, Cat #30-002-CI).

For tumor cell injection, each animal was weighed, and then injectedsubcutaneously in the left and right flank area with 0.1 ml of 1.0×10₈of MDA-MB-453 human breast cancer cells per mL in 50% Matrigel (Corning,Cat #354234) with 25-gauge needle. Cell viability was determined withVi-CELL cell viability analyzer, and only cells with over 95% viabilitywere used for this in vivo study.

For haNK003 cell culture, haNK003 cells were cultured in X-Vivo10 medium(Lonza, Cat # BE02-055Q) supplemented with 5% heat inactivated human ABserum (Innovative Research, Cat # IPLA-SERAB-HI), 100 U penicillin/mland 100 μg/ml streptomycin.

For irradiation, haNK003 cells growing in an exponential growth phasewere harvested and counted for viable cell number and viability. On theappropriate day, the haNK003 cells were irradiated with a dose of 1000cGy using JL Shephard Mark 1 Model 68 ₁₃₇Cs irradiator (service providedby the Department of Radiation Oncology, the University of California,Irvine, Calif. 92697).

For cell preparation for dosing, Irradiated haNK003 cells were kept onice during the transportation to animal facility (1124 W. Carson Street,Torrance, Calif., 90502). Cells were washed twice with cold PBS, andthen were re-suspended in appropriate amount of cold PBS and passedthrough 40 μm cell strainer (Corning, Cat #431750) to make single cellpreparation with a final cell density of 1.25×10₇ or 5×10₇ cells/ml,respectively. Then these irradiated haNK003 cells were stored at RT forIV dosing for animals in an appropriate Group, respectively.

12 NSG mice were selected and randomly assigned to 3 study groups with 4mice per group based on appropriate tumor sizes. Randomization was basedon total tumor volume for each animal and animal body weight. For thisefficacy study, once the average size of tumors reached about 100 mm₃,randomization was conducted and treatment was initiated.

The dosing volume was 200 μl, regardless of an individual animal bodyweight. The dosing route was IV injection via tail vein, and the dosingschedule was twice weekly for total 4 weeks, as indicated in the studyprotocol. On an appropriate day, each animal in Groups F and G received2.5×10₆ and 1×10₇ irradiated haNK003 cells in 200 μl PBS, respectively.The dosing volume was 200 μl, the dosing route was IV injection via tailvein, and the dosing schedule was twice weekly for total 4 weeks.

Animals were observed once daily for general appearance. ClinicalObservations were conducted twice daily and recorded. Animals wereroutinely monitored after treatment for effects on normal behavior suchas mobility, food and water consumption (by visual estimation), and bodyweight (gain/loss).

Tumor size was measured twice weekly in three dimensions using a digitalhand held caliper (once tumor emerges) prior to the first dosing andthen twice weekly prior to euthanasia. The major endpoint was inhibitionor reduction of tumor growth. The tumor volume was expressed in mm₃using the formula: V=0.5×L×W×H where L, W and H are the length, widthand height of the tumor, respectively. The tumor volume was then usedfor calculations of T/C values. T/C (%)=ΔT/ΔC×100, where the ΔT and ΔCare the changes in the mean tumor volumes between an observation day andthe first day of measurement for the treatment and control groups,respectively.

Summary statistics, including mean and standard error of the mean (SEM),were provided for the tumor volume or animal body weight of each groupat each time point. Statistical analyses of difference in tumor volumeor animal body weight change among the groups were evaluated usingtwo-way ANOVA with repeated measures followed by Bonferroni test. Allthe data were analyzed using GraphPad Prism software version 5. p<0.05was considered to be statistically significant.

Results

Data on the antitumor activity of haNK003 as a single agent ins.c.MDA-MB-453 HER2− positive human breast carcinoma xenograft in femaleNSG mice is presented. A summary of the results is tabulated in Table11. haNK003 cells, administered intravenously as a single agent at thedose of 2.5×10⁶ or 1.0×10⁷ cells twice weekly for 4 weeks, significantlyinhibited tumor growth with T/C value of 17.4% and 1.3% (p=0.043 andp=0.006, compared to PBS Group), respectively, as shown in Table 11,FIG. 19. HaNK003 at the dose of 2.5×10⁶ or 1.0×10⁷ cells was welltolerated with a maximum average body weight loss of 0.6% and 5.6%(p=0.203 and p=0.085, compared to PBS Group), respectively, as indicatedin Table 11, FIG. 20. There was not any haNK003 treatment relatedmortality that occurred in this study, as summarized in Table 11.

TABLE 11 Anti-tumor activity of haNK003 cells in MDA-MB-453 human breastcarcinoma s.c. xenograft model in female NSG mice. Animal T/C^(a)MWL^(a) Mortality^(c) Group No. Treatment Dose (%) P value^(b) (%)(n/total) A 4 PBS / / / / 0/4 F 4 Irradiated 2.5 × 10⁶ cells 17.4 P =0.043 0.6 0/4 haNK003 G 4 Irradiated  1 × 10⁷ cells 1.3 P = 0.006 5.60/4 haNK003 Note: ^(a)T/C (%) was calculated using the formula: T/C (%)= ΔT/ΔC × 100, where the ΔT and ΔC are the changes in the mean tumorvolumes between day 26 and the first day of measurement for thetreatment and control groups, respectively. ^(b)MWL: maximum animal bodyweight loss. ^(c)P-value (two-way ANOVA with repeated measures followedby Bonferroni test) vs. PBS treatment Group.

Irradiated haNK003 cells as a single agent at the dose of 2.5×10⁶ or1.0×10⁷ cells significantly inhibited tumor growth in MDA-MB-453 HER2−positive human breast carcinoma xenograft model in female NSG.Irradiated haNK003 cells at both doses were well-tolerated. There wasnot any haNK003 treatment related mortality that occurred.

Example 8. Expression of Genes Associated with Hypoxia is not Reduced inhaNK Cells

Natural Killer (NK) cell lytic activity is suppressed in hypoxicenvironments in vitro (1% O₂) and is associated with downregulation ofNKG2D, perforin and granzyme. There is some variability with NKsensitivity to hypoxia (1% O₂) from normal donors. However, NK celllytic activity can be partially rescued by exogenous IL-2 activation invitro (16 h, 1000 IU/ml). Further, NK cells retain ADCC capacity atunder 1% oxygen conditions.

To determine whether hypoxic conditions alters gene expression in haNKcells, RNA expression was determined in 3 normal donor NK cellpopulations and haNK cells exposed for 5 hours to 20% or 0% O₂. ThreePatient Donors NK cell populations (950, 962, 996) were compared to haNKcells under two conditions, 0% oxygen and 20% oxygen. Pairwise sampleclustering reveals distinct clusters separating haNK from donor NK cells(See FIG. 21). One patient sample appears to have very little change inexpression under hypoxic or pre-hypoxic conditions. Specifically,expression in 962 under 20% oxygen conditions looks very similar to 9960% oxygen and 962 0% oxygen conditions. See FIG. 21. The genesexhibiting the most variability across the samples are shown in FIG. 22.The genes exhibiting the most change between 20% oxygen conditions and0% oxygen conditions are shown in FIG. 23. Genes associated with hypoxiashowing no change in expression in haNK cells between 20% oxygenconditions and 0% oxygen conditions are shown in FIG. 24. These samegenes associated with hypoxia are shown to have reduced expression in950, 962, and 996 samples albeit less so for the 950 sample. See FIG.24.

Example 9. CD16 Expression is More Stable in hank003 Cells

It is desirable to have stable expression of CD16 for endowing superiorcytotoxicity by serial killing of the target cells during antibodydependent cellular toxicity (ADCC). HaNK-003 (with ER-IL-2) expressesCD16 at a high level following activation with PMA (phorbol-12-myristate13-acetate) or upon stimulation with K562 target cells compared toperipheral blood NK cells (donor NK cell). Further CD16 levels inhank003 cells were not considerably affected during and after ADCC bymeasuring CD16 levels by f low cytometry, following the ADCC.

It is known that PMA/ionomycin activation in NK cells leads toactivation of CD16-specific protease and cleavage of CD16, resulting indownregulation of CD16 expression level in the NK cells. To determinethe effect of PMA/ionomycin, both haNK003 cells and donor NK cells werecontacted with 40 nM PMA and 669 nM ionomycin for 1 hour and thenchecked for CD16 expression level. The PMA/ionomycin treatment resultedin 94.36%±3.00 downregulation of CD16 expression in donor NK cells,whereas in haNK003 cells, the treatment resulted in only 30%±0.04down-regulation, i.e. three fold less CD16 down regulation than in donorNK cells (FIG. 25).

It is also known that co-culturing of NK cells with K562 cellsstimulates the CD16 cleavage protease which leads to shedding of CD16surface expression in NK cells. Therefore, both the donor NK cells andhaNK003 cells were cultured with K562 cells and then measured for CD16expression after 4 hours of co-culture. Normal co-culture conditions ofhaNK003 cells with K562 (effectors:targets=1:1) leads to completecytotoxic killing of target cells within 4 hours. CD16 expression wasmeasured again after another 24 hours to allow for recovery of CD16expression in haNK003 cells and donor NK cells to determine thepercentage of CD16 recovery in haNK003 cells and donor NK cells. It wasobserved that CD16 expression levels were down regulated by 60.25%±09 indonor NK cells after 4 hours of co-culture. However, in hank003 cellsCD16 expression was down regulated by only 4.9%±2.57. After 24 hours,the downregulation of CD16 in donor NK cells was 57.54%±26.82, whereasin haNK003 cells it was only 2.78%±3.5, i.e. close to the original CD16level (FIG. 26).

CD16 expression level in haNK003 cells was also measured afterantibody-dependent cell-mediated cytotoxicity (ADCC). ADCC was performedby incubating haNK003 cells with DOHH-2 (CD20+ human lymphoma B-cellline) in the presence of Rituximab (CD20-directed cytolytic monoclonalantibody) followed by measurement of CD16 expression. After ADCC, CD16expression was down regulated by less than 10% in haNK003 cells (FIGS.27A and 27B). The presence of high levels of CD16 even after ADCCindicated that CD16 expression in haNK003 cells is highly stable.

What is claimed is:
 1. A method of treating cancer in a subjectcomprising the steps of: (a) administering to the subject a populationof modified NK-92 cells having antibody-dependent cell-mediatedcytotoxicity (ADCC), wherein the population of modified NK-92 cellscomprise heterologous nucleic acid molecules comprising a nucleic acidsequence with at least 90% identity to CD16 (SEQ ID NO:3) and a nucleicacid sequence with at least 90% identity to IL-2 (SEQ ID NO:5), whereingreater than 90% of the cells in the population of cells express CD56,CD16, CD54, and NKp30 and less than 5% of the cells in the population ofcells express CD3, and wherein the nucleic acid molecules comprise from5′ to 3′ a sequence encoding CD16, an IRES sequence, and a sequenceencoding IL-2; and (b) administering to the subject an antibody, whereinthe administering treats the cancer in the subject.
 2. The method ofclaim 1, wherein the nucleic acid molecules are DNA molecules.
 3. Themethod of claim 1, wherein the cells comprise SEQ ID NO:1 on chromosome17.
 4. The method of claim 1, wherein the mean doubling time of thecells is between 55 and 70 hours.
 5. The method of claim 1, wherein thepopulation of cells maintains the mean doubling time from 1, 2, 3, 4, 5,10, 15, 20, or 25 days.
 6. The method of claim 1, wherein the populationof cells can be passaged every 1, 2, 3, or 4 days.
 7. The method ofclaim 1, wherein the cells are capable of secreting IL-2 at aconcentration of 10 to 60 pg/hour per million cells.
 8. The method ofclaim 1, wherein the cells are irradiated cells.
 9. The method of claim1, wherein the cells have reduced downregulation of expression of CD16compared to a control.
 10. The method of claim 1, wherein the cellsmaintain higher levels of CD16 after ADCC compared to a control.
 11. Themethod of claim 1, wherein the antibody is a monoclonal antibody. 12.The method of claim 1, wherein the cancer is a cancer of the brain,breast, cervix, colon, head & neck, liver, kidney, lung, non-small celllung, or stomach.
 13. The method of claim 1, wherein the population ofcells and the antibody are administered separately.
 14. The method ofclaim 1, wherein the population of cells and the antibody areadministered in the same formulation.
 15. The method of claim 1, whereinthe population of cells and the antibody are administered in separateformulations.
 16. The method of claim 1, wherein the antibody isselected from the group consisting of alemtuzumab, bevacizumab,ibritumomab tiuxetan, ofatumumab, rituximab, and trastuzumab.
 17. Themethod of claim 1, wherein the population of cells and the antibody areadministered on the same day.
 18. The method of claim 1, wherein thedose of the population of cells administered to the subject is between1×10⁹ and 1×10¹⁰ per m².
 19. The method of claim 18, wherein multipledoses of the population of cells are administered to the subject.